Scytovirins and related conjugates, fusion proteins, nucleic acids, vectors, host cells, compositions, antibodies and methods of using scytovirins

ABSTRACT

An isolated or purified antiviral protein of SEQ ID NO: 1, nucleic acids encoding the antiviral protein, cells comprising the nucleic acids, and methods of inhibiting viral infection comprising contacting the virus with the antiviral protein.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This application is the U.S. national phase of International PatentApplication No. PCT/US03/13991 filed on May 15, 2003, which claims tothe benefit of U.S. Provisional Application No. 60/381,322 filed on May16, 2002.

FIELD OF THE INVENTION

The present invention relates to antiviral scytovirins, fusion proteinsand conjugates thereof, compositions comprising same and uses thereof toinhibit viral infections. The invention also relates to nucleic acids,vectors, host cells, compositions thereof, and methods of use thereof toinhibit viral infections. The invention further relates to antibodies.

BACKGROUND OF THE INVENTION

Viral infections remain among the most formidable causes of human andnon-human animal morbidity and mortality worldwide. Effectivepreventions or therapies against most viral pathogens remain elusive.One of the most contemporary and catastrophic examples is the stillrapidly expanding and pervasive worldwide pandemic of HIV (humanimmunodeficiency virus) infection and AIDS (acquired immune deficiencysyndrome). Despite more than two decades of research to find effectivepreventative or therapeutic vaccines or drugs, surprisingly littleprogress has been made. The need for new effective preventative andtherapeutic agents for HIV/AIDS and other potentially lethal viraldiseases remains an urgent global priority.

Most efforts thus far to discover and develop new antiviral prophylacticor therapeutic drugs have focused on classical, non-peptidic “smallmolecules.” For example, nucleoside derivatives, such as AZT, whichinhibit the retroviral reverse transcriptase, were among the firstclinically active agents available commercially for anti-HIV therapy.Although very useful in some patients, the utility of AZT and otheravailable anti-HIV drugs is limited by toxicity and insufficienttherapeutic indices for fully adequate therapy. Also, given the dynamicsof HIV infection (Coffin, Science 267: 483-489 (1995); and Cohen,Science 267: 179 (1995)), it has become increasingly apparent thatagents acting as early as possible in the viral replicative cycle areneeded to inhibit infection of newly produced, uninfected immune cellsgenerated in the body in response to the virus-induced killing ofinfected cells. Also, it is essential to neutralize or inhibit newinfectious virus produced by infected cells. Preferably, new agents,which act directly on the virus and/or upon the early viral host-cellinteractions, to prevent virus/cell attachment and/or fusion and entryof virus into the cell are needed.

Peptidic or proteinaceous agents have historically been shunned in mostdrug discovery and development programs, typically based upon biasedconsiderations of physicochemical properties, in vivo absorption anddisposition, immunogenicity, and the like. However, in recent years,such biases have begun to sway, due to the increasing realization thatthe perceived problems can be circumvented, and that peptidic moleculesoffer tremendous structural diversity that may be exploited fordevelopment of novel therapeutics and preventions of many differentkinds of diseases. Indeed, the foundation of the biotechnology industryis built substantially upon the potential of peptide- and protein-basedtherapeutics.

For example, in the field of HIV therapeutics a novel “rationally”constructed peptide molecule known as T-20 (Kilby, Nat. Med. 4:1302-1307 (1998)) has been recently shown to be a potent inhibitor ofHIV/cell fusion. Early clinical trials of T-20 are revealingconsiderable promise for inhibiting HIV infection in vivo (Cammack,Curr. Opin. Infect. Dis. 14: 13-16 (2001)). Thus, a distinct legitimacyis emerging in the HIV field for further exploration of peptide- andprotein-based prevention and therapeutics of HIV infection and disease.Further reinforcing this momentum is the increasing realization thatnaturally occurring, non-mammalian peptides and proteins may offerentirely unanticipated new avenues for antiviral discovery anddevelopment. An outstanding example is the remarkable HIV-inactivatingprotein cyanovirin-N (Boyd et al., Antimicrob. Agents Chemother. 41:1521-1530 (1997)). This agent is currently the subject of several majorantiviral development programs in the United States under federalauspices, as well as elsewhere within the commercial sector. Clearly,there is great untapped potential for discovery and development ofnovel, non-mammalian antiviral peptides and proteins for unprecedenteduses in prevention and therapeutics of viral diseases.

Accordingly, it is an object of the present invention to provide newantiviral peptides and proteins, as well as fusion proteins andconjugates thereof, and compositions comprising same, and methods ofusing same to inhibit viral infections. It is also an object of thepresent invention to provide nucleic acids, vectors, host cells, andrelated compositions and methods of use thereof to inhibit viralinfections. It is yet another object of the present invention to provideantibodies. These and other objects and advantages of the presentinvention, as well as additional inventive features, will becomeapparent from the description provided herein.

BRIEF SUMMARY OF THE INVENTION

The present invention provides an isolated or purified antiviral proteinconsisting essentially of the amino acid sequence of SEQ ID NO: 1, anamino acid sequence that is about 90% or more identical to SEQ ID NO: 1,an amino acid sequence that is about 90% or more homologous to SEQ IDNO: 1, or an antiviral fragment of any of the foregoing. Also providedis a variant of the isolated or purified antiviral protein, whichcomprises (i) one or more conservative or neutral amino acidsubstitutions and/or (ii) 1, 2 or 3 amino acid additions at theN-terminus and/or C-terminus, with the proviso that the variant hasantiviral activity characteristic of the antiviral protein, whichconsists essentially of the amino acid sequence of SEQ ID NO: 1 andwhich is isolated or purified from Scytonema varium, to a greater orlesser extent but not negated. Similarly provided are a fusion proteinof the antiviral protein or variant thereof and a conjugate of theantiviral protein or variant thereof and at least one effectorcomponent. A composition comprising (i) at least one of the foregoingand (ii) a carrier, excipient or adjuvant therefore is also provided.

The present invention further provides an isolated or purified nucleicacid consisting essentially of a nucleotide sequence encoding the aminoacid sequence of SEQ ID NO: 1, an amino acid sequence that is about 90%or more identical to SEQ ID NO: 1, an amino acid sequence that is about90% or more homologous to SEQ ID NO: 1, or an antiviral fragment of anyof the foregoing, optionally in the form of a vector. Also provided is avariant of the isolated or purified nucleic acid, which comprisesnucleotides encoding (i) one or more conservative or neutral amino acidsubstitutions and/or (ii) up to 1, 2 or 3 amino acid additions at theN-terminus and/or C-terminus, with the proviso that the encoded aminoacid sequence has antiviral activity characteristic of the antiviralprotein, which consists essentially of the amino acid sequence of SEQ IDNO: 1 and which is isolated or purified from Scytonema varium, to agreater or lesser extent but not negated, optionally in the form of avector. Similarly provided is an isolated or purified nucleic acidconsisting essentially of a nucleotide sequence encoding a fusionprotein comprising the antiviral protein, optionally in the form of avector.

Still further provided by the present invention is an isolated cellcomprising an above-described isolated or purified nucleic acid.

A composition comprising (i) an above-described isolated or purifiednucleic acid or variant thereof, optionally as part of an encoded fusionprotein and/or in the form of a vector, and (ii) a carrier, excipient oradjuvant therefore is also provided.

A method of inhibiting a viral infection of a host is further provided.The method comprises administering a viral infection-inhibiting amountof at least one of the following:

-   -   (i) an isolated or purified antiviral protein consisting        essentially of the amino acid sequence of SEQ ID NO: 1, an amino        acid sequence that is about 90% or more identical to SEQ ID NO:        1, an amino acid sequence that is about 90% or more homologous        to SEQ ID NO: 1, or an antiviral fragment of any of the        foregoing,    -   (ii) a variant of (i), which comprises (a) one or more        conservative or neutral amino acid substitutions and/or (b) 1, 2        or 3 amino acid additions at the N-terminus and/or C-terminus,        with the proviso that the variant has antiviral activity        characteristic of the antiviral protein, which consists        essentially of the amino acid sequence of SEQ ID NO: 1 and which        is isolated or purified from Scytonema varium, to a greater or        lesser extent but not negated,    -   (iii) a fusion protein of (i),    -   (iv) a fusion protein of (ii),    -   (v) a conjugate comprising (i) and at least one effector        component,    -   (vi) a conjugate comprising (ii) and at least one effector        component,    -   (vii) a composition comprising one or more of (i)-(vi),    -   (viii) an isolated or purified nucleic acid consisting        essentially of a nucleotide sequence encoding the amino acid        sequence of SEQ ID NO: 1, an amino acid sequence that is about        90% or more identical to SEQ ID NO: 1, an amino acid sequence        that is about 90% or more homologous to SEQ ID NO: 1, or an        antiviral fragment of any of the foregoing, optionally in the        form of a vector,    -   (ix) a variant of (viii), which comprises nucleotides        encoding (a) one or more conservative or neutral amino acid        substitutions and/or (b) up to 1, 2 or 3 amino acid additions at        the N-terminus and/or C-terminus, with the proviso that the        encoded amino acid sequence has antiviral activity        characteristic of the antiviral protein, which consists        essentially of the amino acid sequence of SEQ ID NO: 1 and which        is isolated or purified from Scytonema varium, optionally in the        form of a vector,    -   (x) an isolated or purified nucleic acid consisting essentially        of a nucleotide sequence encoding a fusion protein of (viii),        optionally in the form of a vector,    -   (xi) an isolated or purified nucleic acid consisting essentially        of a nucleotide sequence encoding a fusion protein of (ix),        optionally in the form of a vector,    -   (xii) a composition comprising one or more of (viii)-(xi), and    -   (xiii) an isolated cell comprising (viii), (ix), (x), or (xi).        The method optionally further comprises the prior, simultaneous        or subsequent administration, by the same route or a different        route, of an antiviral agent or another agent that is        efficacious in inhibiting the viral infection.

Still further provided is a method of inhibiting a virus in a biologicalsample or in/on an inanimate object. The method comprises contacting thebiological sample or the inanimate object with a viral-inhibiting amountof at least one of the following:

-   -   (i) an isolated or purified antiviral protein consisting        essentially of the amino acid sequence of SEQ ID NO: 1, an amino        acid sequence that is about 90% or more identical to SEQ ID NO:        1, an amino acid sequence that is about 90% or more homologous        to SEQ ID NO: 1, or an antiviral fragment of any of the        foregoing,    -   (ii) a variant of (i), which comprises (a) one or more        conservative or neutral amino acid substitutions and/or (b) 1, 2        or 3 amino acid additions at the N-terminus and/or C-terminus,        with the proviso that the variant has antiviral activity        characteristic of the antiviral protein, which consists        essentially of the amino acid sequence of SEQ ID NO: 1 and which        is isolated or purified from Scytonema varium, to a greater or        lesser extent but not negated,    -   (iii) a fusion protein of (i),    -   (iv) a fusion protein of (ii),    -   (v) a conjugate comprising (i) and at least one effector        component,    -   (vi) a conjugate comprising (ii) and at least one effector        component, and    -   (vii) a composition comprising one or more of (i)-(vi). The        method optionally further comprises the prior, simultaneous or        subsequent contacting, in the same manner or in different        manner, of the biological sample or inanimate object with an        antiviral agent or another agent that is efficacious in        inhibiting the virus.

Yet still further provided is an antibody to scytovirin, ananti-scytovirin antibody, and a composition comprising same.

A method of inhibiting infection of a mammal with a virus is even stillfurther provided. The method comprises administering to the mammal ananti-scytovirin antibody, or a composition comprising same, in an amountsufficient to induce in the mammal an immune response to the virus. Themethod optionally further comprises the prior, simultaneous orsubsequent administration, by the same or a different route, of anantiviral agent or another agent that is efficacious in inducing animmune response to the virus.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the primary amino acid sequence (SEQ ID NO:1) ofscytovirin. The protein was sequenced by a combination of N-terminalEdman degradation and ESI-MS (electrospray ionization mass spectrometry)of overlapping peptide fragments generated by endoproteinase digestions.Selected peptides isolated by C₁₈ HPLC (high pressure liquidchromatography) from digests with endoproteinases Arg-C and Glu-C areshown. Disulfide cross-links were determined by ESI-MS analysis ofpeptide fragments generated by tryptic digestion of scytovirin, and aremarked (solid lines) above the sequence.

FIG. 2 compares the amino acid sequences of domains 1-48 and 49-95 ofscytovirin (SEQ ID NO: 1). Sequence identities are indicated by verticallines, and conserved changes by colons.

FIG. 3 aligns the amino acid sequence of scytovirin (SEQ ID NO: 1) andthe homologous region of a cloned polypeptide (CL-B) (SEQ ID NO: 2) fromVolvox carteri. Numbers to the top and bottom of the sequences indicateamino acid residue numbers. Sequence identities are indicated byvertical lines, conserved changes by colons, and gaps by dashes.

FIG. 4 aligns amino acid sequences of scytovirin (SEQ ID NO: 3) andchitin-binding domains of select lectins, namely Urtica dioicaagglutinin (UDA; SEQ ID NO: 4), hevein from Hevea brasiliensis (SEQ IDNO: 5), Ac-AMP2 from Amaranthus caudatus (SEQ ID NO: 6), and wheat germagglutinin (WGA) from Triticum aestivum (SEQ ID NO: 7). Conservedresidues are in blocks. The disulfide linkage pattern is indicated belowthe domains for the lectins, and above for scytovirin.

DETAILED DESCRIPTION OF THE INVENTION

The principal overall objective of the present invention is to provideantiviral proteins and fragments thereof, as well as fusion proteins andconjugates comprising same, and broad medical uses thereof, includingprophylactic and/or therapeutic applications against viruses. An initialobservation, which led to the present invention, was antiviral activityin certain extracts from cultured cyanobacteria (blue-green algae)tested in an anti-HIV screen. The screen is one that was conceived in1986 (by M. R. Boyd of the National Institutes of Health) and has beendeveloped and operated at the U.S. National Cancer Institute (NCI) since1988 (see Boyd, in AIDS, Etiology, Diagnosis, Treatment and Prevention,DeVita et al., eds., Philadelphia: Lippincott, 1988, pp. 305-317).

Cyanobacteria (blue-green algae) were specifically chosen for anti-HIVscreening because they had been known to produce a wide variety ofstructurally unique and biologically active non-nitrogenous and aminoacid-derived natural products (Faulkner, Nat. Prod. Rep. 11: 355-394(1994); and Glombitza et al., in Algal and Cyanobacterial Biotechnology,Cresswell, R. C., et al., eds., (1989), pp. 211-218). Thesephotosynthetic prokaryotic organisms are significant producers of cyclicand linear peptides (molecular weight generally <3 kDa), which oftenexhibit hepatotoxic or antimicrobial properties (Okino et al.,Tetrahedron Lett. 34: 501-504 (1993); Krishnamurthy et al., PNAS USA 86:770-774 (1989); Sivonen et al., Chem. Res. Toxicol. 5: 464-469 (1992);Carter et al., J. Org. Chem. 49: 236-241 (1984); and Frankmolle et al.,J. Antibiot. 45: 1451-1457 (1992)). Sequencing studies of highermolecular weight cyanobacterial peptides and proteins have generallyfocused on those associated with primary metabolic processes or onesthat can serve as phylogenetic markers (Suter et al., FEBS Lett. 217:279-282 (1987); Rumbeli et al., FEBS Lett. 221: 1-2 (1987); Swanson etal., J. Biol. Chem. 267: 16146-16154 (1992); Michalowski et al., NucleicAcids Res. 18: 2186 (1990); Sherman et al., in The Cyanobacteria, Fay etal., eds., Elsevier: New York (1987), pp. 1-33; and Rogers, in TheCyanobacteria, Fay et al., eds., Elsevier: New York (1987), pp. 35-67).The first example of a potent antiviral protein, particularly ananti-HIV protein, from a cyanobacterium was cyanovirin-N (Boyd et al.,Antimicrob. Agents Chemother. 41: 1521-1530 (1997)) from Nostocellipsosporum. Otherwise, in general, proteins with antiviral propertieshave not been associated with cyanobacterial sources.

The cyanobacterial extract leading to the present invention was amongmany thousands of different extracts initially selected randomly andtested blindly in the anti-HIV screen described above. A number of theseextracts had been determined preliminarily to show anti-HIV activity inthe NCI screen (Patterson et al., J. Phycol. 29: 125-130 (1993)). Fromthis group, an aqueous extract from Scytonema varium, which had beenprepared as described (Patterson (1993), supra) and which showed anunusually high anti-HIV potency and in vitro “therapeutic index” in theNCI primary screen, was selected for detailed investigation. A specificbioassay-guided strategy was used to isolate and purify a homogenousprotein highly active against HIV.

In the bioassay-guided strategy, initial selection of the extract forfractionation, as well as the decisions concerning the overall chemicalisolation method to be applied, and the nature of the individual stepstherein, are determined by interpretation of biological testing data.The anti-HIV screening assay (e.g., see Boyd (1988), supra; and Weislowet al., J. Natl. Cancer Inst. 81: 577-586 (1989)), which is used toguide the isolation and purification process, measures the degree ofprotection of human T-lymphoblastoid cells from the cytopathic effectsof HIV. Fractions of the extract of interest are prepared using avariety of chemical means and are tested blindly in the primary screen.Active fractions are separated further, and the resulting subfractionsare likewise tested blindly in the screen. This process is repeated asmany times as necessary in order to obtain the active compound(s), i.e.,antiviral fraction(s) representing pure compound(s), which then can besubjected to detailed chemical analysis and structural elucidation.Using this strategy, aqueous extracts of Scytonema varium were shown tocontain an antiviral protein. The present invention describes morespecifically a method of obtaining a wild-type scytovirin from Scytonemavarium. Such a method comprises (a) identifying an extract of Scytonemavarium containing antiviral activity, (b) optionally removing highmolecular weight biopolymers from the extract, (c) antiviralbioassay-guided fractionating of the extract to obtain a crude extractof scytovirin, and (d) purifying the crude extract by reverse-phase HPLCto obtain a scytovirin (see, also, Example 1). More specifically, themethod involves the use of an anti-HIV bioassay to guide fractionationof the extract.

A natural, wild-type scytovirin (a protein of exactly SEQ ID NO:1),which was isolated and purified as described in more detail in Example1, was subjected to conventional procedures typically used to determinethe amino acid sequence of a given pure protein. Thus, the scytovirinwas initially sequenced by N-terminal Edman degradation of intactprotein and numerous overlapping peptide fragments generated byendoproteinase digestion. ESI-MS of reduced, HPLC-purified, naturalscytovirin showed a molecular ion consistent with the calculated value.These studies indicated that the wild-type scytovirin from Scytonemavarium was comprised of a unique sequence of 95 amino acids havinginternal sequence homology, but minimal overall homology, to previouslydescribed proteins or transcription products of known nucleotidesequences (see Example 2 and FIGS. 1-5). No more than about 55% homologyfrom wild-type scytovirin was found in any amino acid sequences orsubsequences from known proteins. Given the chemically deduced aminoacid sequence of wild-type scytovirin, a corresponding recombinantscytovirin can be readily created by one ordinarily skilled in the art(e.g., see below) and can be used to demonstrate further that thededuced amino acid sequence is, indeed, active against a virus, such asHIV. One skilled in the art will appreciate that functional (e.g.,antiviral) scytovirin homologs can be obtained from the natural sourceor can be recombinantly produced.

Accordingly, the present invention provides an isolated or purifiedantiviral protein consisting essentially of the amino acid sequence ofSEQ ID NO: 1, an amino acid sequence that is about 60%, 65%, 70%, 75%,80%, 85% or 90% or more homologous to SEQ ID NO: 1, an amino acidsequence that is about 60%, 65%, 70%, 75%, 80%, 85% or 90% or moreidentical to SEQ ID NO: 1 (in which a letter indicates the standardamino acid designated by that letter; the amino acid sequence is givenfrom left to right and top to bottom, such that the first amino acid isamino-terminal and the last amino acid is carboxyl-terminal), or anantiviral fragment of any of the foregoing. The protein preferablycomprises an amino end and a carboxyl end. The protein can compriseD-amino acids, L-amino acids or a mixture of D- and L-amino acids. TheD-form of the amino acids, however, is particularly preferred, since aprotein comprised of D-amino acids is expected to have a greaterretention of its biological activity in vivo, given that the D-aminoacids are not recognized by naturally occurring proteases.

The term “isolated” as used herein means having been removed from itsnatural environment. The term “purified” as used herein means havingbeen increased in purity, wherein “purity” is a relative term, and notto be construed as absolute purity. By “antiviral” is meant that theprotein or fragment thereof can inhibit a virus, in particular aretrovirus, specifically a primate immunodeficiency virus, morespecifically a human immunodeficiency virus (HIV), such as HIV-1, HIV-2or SIV.

Preferably, the antiviral protein or fragment thereof is isolated orpurified from Scytonema varium. Accordingly, the terms “scytovirin” and“scytovirins” are used herein generically to refer to an isolated orpurified protein consisting essentially of SEQ ID NO: 1, as well asantiviral fragments thereof, whether isolated or purified from nature,recombinantly produced, or synthesized, and substantially identical orhomologous proteins (as defined herein). An antiviral fragment can begenerated, for example, by removing 1-20, preferably 1-10, morepreferably 1, 2, 3, 4, or 5, and most preferably 1 or 2, amino acidsfrom one or both ends, preferably from only one end, and most preferablyfrom the amino-terminal end, of the wild-type scytovirin, such aswild-type scytovirin of SEQ ID NO: 1.

In view of the foregoing, the present invention provides a variant of anisolated or purified antiviral protein, wherein the variant comprises(i) one or more conservative or neutral amino acid substitutions and/or(ii) 1-20, preferably 1-10, more preferably 1, 2, 3, 4 or 5, and evenmore preferably, 1, 2, or 3, amino acid additions at the N-terminusand/or the C-terminus, with the proviso that the variant has antiviralactivity characteristic of the antiviral protein, which consistsessentially of the amino acid sequence of SEQ ID NO: 1 and which isisolated or purified from Scytonema varium, to a greater or lesserextent but not negated.

Alterations of the native amino acid sequence to produce variantproteins can be done by a variety of means known to those skilled in theart. For instance, amino acid substitutions can be convenientlyintroduced into the proteins at the time of synthesis. Alternatively,site-specific mutations can be introduced by ligating into an expressionvector a synthesized oligonucleotide comprising the modified site.Alternately, oligonucleotide-directed, site-specific mutagenesisprocedures can be used, such as disclosed in Walder et al., Gene 42: 133(1986); Bauer et al., Gene 37: 73 (1985); Craik, Biotechniques, 12-19(January 1995); and U.S. Pat. Nos. 4,518,584 and 4,737,462.

It is within the skill of the ordinary artisan to select synthetic andnaturally-occurring amino acids that effect conservative or neutralsubstitutions for any particular naturally-occurring amino acids. Theordinarily skilled artisan desirably will consider the context in whichany particular amino acid substitution is made, in addition toconsidering the hydrophobicity or polarity of the side-chain, thegeneral size of the side chain and the pK value of side-chains withacidic or basic character under physiological conditions. For example,lysine, arginine, and histidine are often suitably substituted for eachother, and more often arginine and histidine. As is known in the art,this is because all three amino acids have basic side chains, whereasthe pK value for the side-chains of lysine and arginine are much closerto each other (about 10 and 12) than to histidine (about 6). Similarly,glycine, alanine, valine, leucine, and isoleucine are often suitablysubstituted for each other, with the proviso that glycine is frequentlynot suitably substituted for the other members of the group. This isbecause each of these amino acids are relatively hydrophobic whenincorporated into a polypeptide, but glycine's lack of an α-carbonallows the phi and psi angles of rotation (around the α-carbon) so muchconformational freedom that glycinyl residues can trigger changes inconformation or secondary structure that do not often occur when theother amino acids are substituted for each other. Other groups of aminoacids frequently suitably substituted for each other include, but arenot limited to, the group consisting of glutamic and aspartic acids; thegroup consisting of phenylalanine, tyrosine and tryptophan; and thegroup consisting of serine, threonine and, optionally, tyrosine.Additionally, the ordinarily skilled artisan can readily group syntheticamino acids with naturally-occurring amino acids.

If desired, the proteins of the invention (including antiviralfragments, variant proteins, fusion proteins, and conjugates) can bemodified, for instance, by glycosylation, amidation, carboxylation, orphosphorylation, or by the creation of acid addition salts, amides,esters, in particular C-terminal esters, and N-acyl derivatives of theproteins of the invention. The proteins also can be modified to createprotein derivatives by forming covalent or noncovalent complexes withother moieties in accordance with methods known in the art.Covalently-bound complexes can be prepared by linking the chemicalmoieties to functional groups on the side chains of amino acidscomprising the proteins, or at the N- or C-terminus. Desirably, suchmodifications and conjugations do not adversely affect the activity ofthe proteins (and variants thereof). While such modifications andconjugations can have greater or lesser activity, the activity desirablyis not negated and is characteristic of the unaltered protein.

The proteins (and fragments, variants and fusion proteins) can beprepared by any of a number of conventional techniques. The protein canbe isolated or purified from a naturally occurring source or from arecombinant source. For instance, in the case of recombinant proteins, aDNA fragment encoding a desired protein can be subcloned into anappropriate vector using well-known molecular genetic techniques (see,e.g., Maniatis et al., Molecular Cloning: A Laboratory Manual 2nd ed.(Cold Spring Harbor Laboratory, 1989) and other references cited hereinunder “EXAMPLES”). The fragment can be transcribed and the proteinsubsequently translated in vitro. Commercially available kits also canbe employed (e.g., such as manufactured by Clontech, Palo Alto, Calif.;Amersham Life Sciences, Inc., Arlington Heights, Ill.; InVitrogen, SanDiego, Calif., and the like). The polymerase chain reaction optionallycan be employed in the manipulation of nucleic acids.

Such proteins also can be synthesized using an automated peptidesynthesizer in accordance with methods known in the art. Alternately,the protein (and fragments, variants, and fusion proteins) can besynthesized using standard peptide synthesizing techniques well-known tothose of skill in the art (e.g., as summarized in Bodanszky, Principlesof Peptide Synthesis, (Springer-Verlag, Heidelberg: 1984)). Inparticular, the protein can be synthesized using the procedure ofsolid-phase synthesis (see, e.g., Merrifield, J. Am. Chem. Soc., 85:2149-54 (1963); Barany et al., Int. J. Peptide Protein Res. 30: 705-739(1987); and U.S. Pat. No. 5,424,398). If desired, this can be done usingan automated peptide synthesizer. Removal of the t-butyloxycarbonyl(t-BOC) or 9-fluorenylmethyloxycarbonyl (Fmoc) amino acid blockinggroups and separation of the protein from the resin can be accomplishedby, for example, acid treatment at reduced temperature. Theprotein-containing mixture then can be extracted, for instance, withdiethyl ether, to remove non-peptidic organic compounds, and thesynthesized protein can be extracted from the resin powder (e.g., withabout 25% w/v acetic acid). Following the synthesis of the protein,further purification (e.g., using HPLC) optionally can be done in orderto eliminate any incomplete proteins, polypeptides, peptides or freeamino acids. Amino acid and/or HPLC analysis can be performed on thesynthesized protein to validate its identity. For other applicationsaccording to the invention, it may be preferable to produce the proteinas part of a larger fusion protein, either by chemical conjugation, orthrough genetic means, such as are known to those skilled in the art. Inthis regard, the present invention also provides a fusion proteincomprising the isolated or purified antiviral protein (or fragmentthereof) or variant thereof and one or more other protein(s) having anydesired properties or effector functions, such as cytotoxic orimmunological properties, or other desired properties, such as tofacilitate isolation, purification or analysis of the fusion protein.Preferably, the fusion protein comprises albumin.

A conjugate comprising (i) the isolated or purified antiviral protein(or fragment thereof) or variant thereof and (ii) at least one effectorcomponent is also provided. Preferably, the at least one effectorcomponent, which can be the same or different, is selected from thegroup consisting of polyethylene glycol, dextran, an immunologicalreagent, a toxin, an antiviral agent, and a solid support matrix.

“Immunological reagent,” for example, may refer to an antibody, animmunoglobulin, or an immunological recognition element. Animmunological recognition element is an element, such as a peptide, forexample a FLAG octapeptide leader sequence, that can be appended to makea recombinant scytovirin-FLAG fusion protein, wherein the FLAG elementfacilitates, through immunological recognition, isolation and/orpurification and/or analysis of the protein (or fragment thereof) orvariant thereof to which it is attached.

A “toxin” can be Pseudomonas exotoxin. An “antiviral agent” can be AZT,ddI, ddC, 3TC gancyclovir, fluorinated dideoxynucleosides, nevirapine,R82913, Ro 31-8959, BI-RJ-70, acyclovir, α-interferon, recombinant sCD4,michellamines, calanolides, nonoxynol-9, gossypol and derivativesthereof, gramicidin, and cyanovirin-N or a functional homolog orderivative thereof. A “solid support matrix” can be a magnetic bead, aflow-through matrix, or a matrix comprising a contraceptive device, suchas a condom, diaphragm, cervical cap, vaginal ring or sponge. In analternative embodiment, a solid support matrix can be an implant forsurgical implantation in a host and later removal.

In view of the foregoing, the present invention further provides acomposition comprising (i) at least one of the isolated or purifiedantiviral protein (or fragment thereof), a variant thereof, a fusionprotein of the antiviral protein (or fragment thereof) or variantthereof, and a conjugate of the antiviral protein (or fragment thereof)or variant thereof, and (ii) a carrier, excipient or adjuvant therefore.Preferably, component (i) of the composition is present in an antiviraleffective amount and the carrier is pharmaceutically acceptable. By“antiviral effective amount” is meant an amount sufficient to inhibitthe infectivity of the virus.

The carrier can be any of those conventionally used and is limited onlyby chemico-physical considerations, such as solubility and lack ofreactivity with the active agent of the present invention, and by theroute of administration. It is preferred that the pharmaceuticallyacceptable carrier be one which is chemically inert to the active agentand one which has no detrimental side effects or toxicity under theconditions of use. The pharmaceutically acceptable carriers describedherein, for example, vehicles, adjuvants, excipients, and diluents, arewell-known to those ordinarily skilled in the art and are readilyavailable to the public. Typically, the composition, such as apharmaceutical composition, can comprise a physiological salinesolution; dextrose or other saccharide solution; or ethylene, propylene,polyethylene, or other glycol.

The present invention also provides an isolated or purified nucleic acidconsisting essentially of a nucleotide sequence encoding the amino acidsequence of SEQ ID NO: 1, an amino acid sequence that is about 60%, 65%,70%, 75%, 80%, 85% or 90% or more identical to SEQ ID NO: 1, an aminoacid sequence that is about 60%, 65%, 70%, 75%, 80%, 85% or 90% or morehomologous to SEQ ID NO: 1, or an antiviral fragment of any of theforegoing, optionally in the form of a vector. The terms “purified” and“isolated” have the meaning set forth above. The term “nucleic acid” asused herein means a polymer of DNA or RNA, (i.e., a polynucleotide),which can be single-stranded or double-stranded, synthesized or obtainedfrom natural sources, and which can contain natural, non-natural oraltered nucleotides.

When the above isolated or purified nucleic acid is characterized interms of “percentage of sequence identity,” a given nucleic acidmolecule as described above is compared to a nucleic acid moleculeencoding a corresponding gene (i.e., the reference sequence) byoptimally aligning the nucleic acid sequences over a comparison window,wherein the portion of the polynucleotide sequence in the comparisonwindow may comprise additions or deletions (i.e., gaps) as compared tothe reference sequence, which does not comprise additions or deletions,for optimal alignment of the two sequences. The percentage of sequenceidentity is calculated by determining the number of positions at whichthe identical nucleic acid base occurs in both sequences, i.e., thenumber of matched positions, dividing the number of matched positions bythe total number of positions in the window of comparison, andmultiplying the result by 100 to yield the percentage of sequenceidentity. Optimal alignment of sequences for comparison may be conductedby computerized implementations of known algorithms (e.g., GAP, BESTFIT,FASTA, and TFASTA in the Wisconsin Genetics Software Package, GeneticsComputer Group (GCG), 575 Science Dr., Madison, Wis., or BlastN andBlastX available from the National Center for Biotechnology Information,Bethesda, Md.), or by inspection. Sequences are typically compared usingBESTFIT or BlastN with default parameters.

“Substantial sequence identity” means that about 60%, preferably about65%, more preferably about 70%, still more preferably about 75%, evenmore preferably about 80%, even still more preferably about 85%, andmost preferably about 90% or more of the sequence of a given nucleicacid molecule is identical to a given reference sequence. Typically, twopolypeptides are considered to be substantially identical if about 60%,preferably about 65%, more preferably about 70%, still more preferablyabout 75%, even more preferably about 80%, even still more preferablyabout 85%, and most preferably about 90% or more of the amino acids ofwhich the polypeptides are comprised are identical to or representconservative substitutions of the amino acids of a given referencesequence.

Another indication that polynucleotide sequences are substantiallyidentical is if two molecules selectively hybridize to each other understringent conditions. The phrase “selectively hybridizing to” refers tothe selective binding of a single-stranded nucleic acid probe to asingle-stranded target DNA or RNA sequence of complementary sequencewhen the target sequence is present in a preparation of heterogeneousDNA and/or RNA. Stringent conditions are sequence-dependent and will bedifferent in different circumstances. Generally, stringent conditionsare selected to be about 20° C. lower than the thermal melting point(Tm) for the specific sequence at a defined ionic strength and pH. TheTm is the temperature (under defined ionic strength and pH) at which 50%of the target sequence hybridizes to a perfectly matched probe.

In view of the above, “stringent conditions” preferably allow up toabout 25% mismatch, more preferably up to about 15% mismatch, and mostpreferably up to about 10% mismatch. “At least moderately stringentconditions” preferably allow for up to about 40% mismatch, morepreferably up to about 30% mismatch, and most preferably up to about 20%mismatch. “Low stringency conditions” preferably allow for up to about60% mismatch, more preferably up to about 50% mismatch, and mostpreferably up to about 40% mismatch. Hybridization and wash conditionsthat result in such levels of stringency can be selected by theordinarily skilled artisan using the references cited under “EXAMPLES”among others.

One of ordinary skill in the art will appreciate, however, that twopolynucleotide sequences can be substantially different at the nucleicacid level, yet encode substantially similar, if not identical, aminoacid sequences, due to the degeneracy of the genetic code. The presentinvention is intended to encompass such polynucleotide sequences.

With respect to the isolated or purified nucleic acid of the presentinvention, it is preferred that no insertions, deletions, inversions,and/or substitutions are present in the nucleic acid. However, it may besuitable in some instances for the isolated or purified nucleic acid toencode one or more conservative and/or neutral amino acid substitutionsand/or amino acid additions at the N-terminus and/or C-terminus. In thisregard, the present invention further provides a variant of theabove-described isolated or purified nucleic acid, wherein the variantcomprises nucleotides encoding (i) one or more conservative or neutralamino acid substitutions and/or (ii) up to 20, preferably up to 10, morepreferably 1, 2, 3, 4 or 5, and even more preferably, 1, 2, or 3, aminoacid additions at the N-terminus and/or the C-terminus, with the provisothat the encoded amino acid sequence has antiviral activitycharacteristic of the antiviral protein, which consists essentially ofthe amino acid sequence of SEQ ID NO: 1 and which is isolated orpurified from Scytonema varium, to a greater or lesser extent but notnegated, optionally in the form of a vector.

A variety of techniques used to synthesize the oligonucleotides of thepresent invention are known in the art. See, for example, Lemaitre etal., PNAS USA 84: 648-652 (1987).

Given the present disclosure, it will be apparent to one ordinarilyskilled in the art that certain modified scytovirin gene sequences willcode for a fully functional, i.e., antiviral, such as anti-HIV,scytovirin homolog. A minimum essential DNA coding sequence(s) for afunctional scytovirin can readily be determined by one skilled in theart, for example, by synthesis and evaluation of sub-sequencescomprising the wild-type scytovirin, and by site-directed mutagenesisstudies of the scytovirin DNA coding sequence.

In view of the above, the present invention also provides a vectorcomprising an above-described isolated or purified nucleic acidmolecule, optionally as part of an encoded fusion protein. The vectorcan be targeted to a cell-surface receptor if so desired. A nucleic acidmolecule as described above can be cloned into any suitable vector andcan be used to transform or transfect any suitable host. The selectionof vectors and methods to construct them are commonly known to personsof ordinary skill in the art and are described in general technicalreferences (see, in general, “Recombinant DNA Part D,” Methods inEnzymology, Vol. 153, Wu and Grossman, eds., Academic Press (1987) andthe references cited herein under “EXAMPLES”). Desirably, the vectorcomprises regulatory sequences, such as transcription and translationinitiation and termination codons, which are specific to the type ofhost (e.g., bacterium, fungus, plant or animal) into which the vector isto be introduced, as appropriate and taking into consideration whetherthe vector is DNA or RNA. Preferably, the vector comprises regulatorysequences that are specific to the genus of the host. Most preferably,the vector comprises regulatory sequences that are specific to thespecies of the host.

Constructs of vectors, which are circular or linear, can be prepared tocontain an entire nucleic acid as described above or a portion thereofligated to a replication system functional in a prokaryotic oreukaryotic host cell. Replication systems can be derived from ColE1, 2mμ plasmid, λ, SV40, bovine papilloma virus, and the like.

In addition to the replication system and the inserted nucleic acid, theconstruct can include one or more marker genes, which allow forselection of transformed or transfected hosts. Marker genes includebiocide resistance, e.g., resistance to antibiotics, heavy metals, etc.,complementation in an auxotrophic host to provide prototrophy, and thelike.

One of ordinary skill in the art will appreciate that any of a number ofvectors known in the art are suitable for use in the invention. Suitablevectors include those designed for propagation and expansion or forexpression or both. Examples of suitable vectors include, for instance,plasmids, plasmid-liposome complexes, and viral vectors, e.g.,parvoviral-based vectors (i.e., adeno-associated virus (AAV)-basedvectors), retroviral vectors, herpes simplex virus (HSV)-based vectors,and adenovirus-based vectors. Any of these expression constructs can beprepared using standard recombinant DNA techniques described in, e.g.,Sambrook et al., Molecular Cloning: A Laboratory Manual, 2^(nd) edition,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989);Ausubel et al., Current Protocols in Molecular Biology, GreenePublishing Associates and John Wiley & Sons, New York, N.Y. (1994);Fischer et al., Transgenic Res. 9(4-5): 279-299 (2000); Fischer et al.,J. Biol. Regul. Homeost. Agents 14: 83-92 (2000); deWilde et al., PlantMolec. Biol. 43: 347-359 (2000); Houdebine, Transgenic Research 9:305-320 (2000); Brink et al., Theriogenology 53: 139-148 (2000); Pollocket al., J. Immunol. Methods 231: 147-157 (1999); Conrad et al., PlantMolec. Biol. 38: 101-109 (1998); Staub et al., Nature Biotech. 18:333-338 (2000); McCormick et al., PNAS USA 96: 703-708 (1999); Zeitlinet al., Nature Biotech. 16: 1361-1364 (1998); Tacker et al., Microbesand Infection 1: 777-783 (1999); and Tacket et al., Nature Med. 4(5):607-609 (1998). Examples of cloning vectors include the pUC series, thepBluescript series (Stratagene, LaJolla, Calif.), the pET series(Novagen, Madison, Wis.), the pGEX series (Pharmacia Biotech, Uppsala,Sweden), and the pEX series (Clonetech, Palo Alto, Calif.).Bacteriophage vectors, such as λGT10, λGT11, λZapII (Stratagene), λEMBL4, and λ NM1149, also can be used. Examples of plant expressionvectors include pBI101, pBI101.2, pBI101.3, pBI121 and pBIN19(Clonetech, Palo Alto, Calif.). Examples of animal expression vectorsinclude pEUK-C1, pMAM and pMAMneo (Clonetech).

An expression vector can comprise a native or normative promoteroperably linked to an isolated or purified nucleic acid as describedabove. The selection of promoters, e.g., strong, weak, inducible,tissue-specific and developmental-specific, is within the skill in theart. Similarly, the combining of a nucleic acid molecule as describedabove with a promoter is also within the skill in the art.

Optionally, the isolated or purified nucleic acid molecule, upon linkagewith another nucleic acid molecule, can encode a fusion protein, such asa fusion protein containing a functional scytovirin component plus afusion component conferring additional desired attribute(s) to thecomposite protein. For example, a fusion sequence for a toxin orimmunological reagent, as defined above, can be added to facilitatepurification and analysis of the functional protein (e.g., such as aFLAG-scytovirin fusion protein) or for specific targeting to a virus orviral-infected cells, e.g., HIV and/or HIV-infected cells. In theseinstances, the scytovirin moiety serves not only as antiviral or as aneutralizing agent but also as a targeting agent to direct the effectoractivities of these molecules selectively against a given virus, such asHIV. Thus, for example, a therapeutic agent can be obtained by combiningthe HIV-targeting function of a functional scytovirin with a toxin aimedat neutralizing infectious virus and/or by destroying cells producinginfectious virus, such as HIV. Similarly, a therapeutic agent can beobtained, which combines the viral-targeting function of a scytovirinwith the multivalency and effector functions of various immunoglobulinsubclasses. Example 4 further illustrates the viral-targeting,specifically viral envelope glycoprotein targeting, properties of ascytovirin.

The generation of fusion proteins is within the ordinary skill in theart (see, e.g., Chaudhary et al. (1988), supra, and the references citedunder “EXAMPLES”) and can involve the use of restriction enzyme orrecombinational cloning techniques (see, e.g., Gateway™ (Invitrogen,Carlsbad, Calif.)). See, also, U.S. Pat. No. 5,314,995. In atranscriptional gene fusion, the DNA or cDNA will contain its owncontrol sequence directing appropriate production of protein (e.g.,ribosome binding site, translation initiation codon, etc.), and thetranscriptional control sequences (e.g., promoter elements and/orenhancers) will be provided by the vector. In a translational genefusion, transcriptional control sequences as well as at least some ofthe translational control sequences (i.e., the translational initiationcodon) will be provided by the vector. In the case of a translationalgene fusion, a chimeric protein will be produced.

Viral-targeted conjugates also can be prepared by chemical coupling ofthe targeting component with an effector component. The most feasible orappropriate technique to be used to construct a given scytovirinconjugate will be selected based upon consideration of thecharacteristics of the particular effector molecule selected forcoupling to a scytovirin. For example, with a selected non-proteinaceouseffector component, chemical coupling may be the only feasible optionfor creating the desired scytovirin conjugate.

Examples of effector components or other functional reagents suitablefor chemical coupling to a scytovirin and thereby used as effectorcomponents in the present inventive conjugates can include, for example,polyethylene glycol, dextran, albumin, a solid support matrix, and thelike, whose intended effector functions may include one or more of thefollowing: to improve stability of the conjugate; to increase thehalf-life of the conjugate; to increase resistance of the conjugate toproteolysis; to decrease the immunogenicity of the conjugate; to providea means to attach or immobilize a functional scytovirin onto a solidsupport matrix (e.g., see, for example, Harris, in Poly(Ethylene Glycol)Chemistry: Biotechnical and Biomedical Applications, Harris, ed., PlenumPress: New York (1992), pp. 1-14); to immobilize the scytovirin (i.e.,in such instance the solid support matrix is an effector component ofthe scytovirin conjugate). Conjugates furthermore can comprise afunctional scytovirin coupled to more than one effector component, eachof which, optionally, can have different effector functions (e.g., suchas a toxin molecule or an immunological reagent, and a polyethyleneglycol or dextran or albumin molecule, and a solid support matrix).Diverse applications and uses of functional proteins and peptides, suchas in the present instance a functional scytovirin, attached to orimmobilized on a solid support matrix, are exemplified more specificallyfor poly(ethylene glycol) conjugated proteins or peptides in a review byHolmberg et al. (In Poly(Ethylene Glycol) Chemistry: Biotechnical andBiomedical Applications, Harris, ed., Plenum Press: New York (1992), pp.303-324). Preferred examples of solid support matrices include magneticbeads, a flow-through matrix, and a matrix comprising a contraceptivedevice, such as a condom, a diaphragm, a cervical cap, a vaginal ring ora sponge.

Example 4 further reveals the specificity of effects of a scytovirin onviral molecular targets, particularly the envelope glycoproteins,especially gp120. Example 5 exemplifies the range of antiviral activityof a representative scytovirin against different CD4⁺-tropicimmunodeficiency virus strains in different target cells. Clinicalisolates and laboratory strains have essentially equivalent sensitivityto the scytovirins. Cocultivation of chronically infected and uninfectedCEM-SS cells with scytovirin causes a concentration-dependent inhibitionof cell-to-cell fusion and virus transmission; similarly, binding andfusion inhibition assays employing HeLa-CD4-LTR-β-galactosidase cellsalso confirm scytovirin inhibition of virus-cell and/or cell-cellbinding.

In addition to using the aforementioned assays, the anti-viral, e.g.,anti-HIV, activity of the scytovirins and conjugates thereof of thepresent invention can be further demonstrated in a series ofinterrelated in vitro antiviral assays (Gulakowski et al., J. Virol.Methods 33: 87-100 (1991), which accurately predict for antiviralactivity in humans. These assays measure the ability of compounds toprevent the replication of HIV and/or the cytopathic effects of HIV onhuman target cells. These measurements directly correlate with thepathogenesis of HIV-induced disease in vivo, and, therefore, establishthe utility of the present invention.

The scytovirins, and antiviral fragments, fusion proteins and conjugatesthereof, of the present invention can be shown to inhibit a virus,specifically a retrovirus, more specifically an immunodeficiency virus,such as the human immunodeficiency virus, i.e., HIV-1 or HIV-2. Thepresent inventive agents can be used to inhibit other retroviruses aswell as other viruses (see, e.g., Principles of Virology: MolecularBiology, Pathogenesis, and Control, Flint et al., eds., ASM Press:Washington, D.C., 2000, particularly Chapter 19). Examples of virusesinclude, but are not limited to, for example, one or more of thefollowing: Type C and Type D retroviruses, HTLV-1, HTLV-2, HIV, FIV,FLV, SIV, MLV, BLV, BIV, equine infectious virus, anemia virus, aviansarcoma viruses, such as Rous sarcoma virus (RSV), hepatitis type A, B,non-A and non-B viruses, arboviruses, varicella viruses, human herpesvirus (e.g., HHV-6), measles, mumps and rubella viruses, pox viruses,influenza viruses A and B, Ebola and other hemorrhagic fever viruses,and other viruses.

Thus, the present invention further provides a composition comprising(i) one or more of an above-described purified or isolated nucleic acidor variant thereof, optionally as part of an encoded fusion protein, and(ii) a carrier, excipient or adjuvant. Preferably, (i) is present in anantiviral effective amount and the composition is pharmaceuticallyacceptable. The composition can further comprise at least one additionalactive agent, such as an antiviral agent other than a scytovirin (orantiviral fragment, fusion protein or conjugate thereof), in anantiviral effective amount. Suitable antiviral agents include AZT, ddA,ddI, ddC, 3TC gancyclovir, fluorinated dideoxynucleosides, acyclovir,α-interferon, nonnucleoside analog compounds, such as nevirapine (Shihet al., PNAS 88: 9878-9882, (1991)), TIBO derivatives, such as R82913(White et al., Antiviral Res. 16: 257-266 (1991)), Ro31-8959, BI-RJ-70(Merigan, Am. J. Med. 90 (Suppl.4A): 8S-17S (1991)), michellamines (Boydet al., J. Med. Chem. 37: 1740-1745 (1994)) and calanolides (Kashman etal., J. Med. Chem. 35: 2735-2743 (1992)), nonoxynol-9, gossypol andderivatives, gramicidin, cyanovirin-N and functional homologs thereof(Boyd et al. (1997), supra). Other exemplary antiviral compounds includeprotease inhibitors (see R. C. Ogden and C. W. Flexner, eds., ProteaseInhibitors in AIDS Therapy, Marcel Dekker, NY, 2001), such as saquinavir(see I. B. Duncan and S. Redshaw, in R. C. Ogden and C. W. Flexner,supra, pp. 27-48), ritonavir (see D. J. Kempf, in R. C. Ogden and C. W.Flexner, supra, pp. 49-64), indinavir (see B. D. Dorsey and J. P. Vacca,in R. C. Ogden and C. W. Flexner, supra, pp. 65-84), nelfinavir (see S.H. Reich, in R. C. Ogden and C. W. Flexner, supra, pp. 85-100),amprenavir (see R. D. Tung, in R. C. Ogden and C. W. Flexner, supra, pp.101-118), and anti-TAT agents. If the composition is to be used toinduce an immune response, it comprises an immune response-inducingamount of the present inventive agent and can further comprise animmunoadjuvant, such as polyphosphazene polyelectrolyte.

The pharmaceutical composition can contain other pharmaceuticals, suchas virucides, immunomodulators, immunostimulants, antibiotics andabsorption enhancers. Exemplary immunomodulators and immunostimulantsinclude various interleukins, sCD4, cytokines, antibody preparations,blood transfusions, and cell transfusions. Exemplary antibiotics includeantifungal agents, antibacterial agents, and anti-Pneumocystitis carniiagents. Exemplary absorption enhancers include bile salts and othersurfactants, saponins, cyclodextrins, and phospholipids (Davis (1992),supra).

An isolated cell comprising an above-described purified or isolatednucleic acid or variant thereof, optionally in the form of a vector,which is optionally targeted to a cell-surface receptor, is alsoprovided. Examples of host cells include, but are not limited to, ahuman cell, a human cell line, E. coli, B. subtilis, P. aerugenosa, S.cerevisiae, and N. crassa. E. coli, in particular E. coli TB-1, TG-2,DH5α, XL-Blue MRF′ (Stratagene), SA2821 and Y1090. Preferably, the cellis a bacterium or yeast. A preferred bacterium is lactobacillus. Theabove-described nucleic acid or variant thereof, optionally in the formof a vector, can be introduced into a host cell using such techniques astransfection, electroporation, transduction, micro-injection,transformation, and the like.

Thus, using an appropriate DNA coding sequence, a recombinant scytovirincan be made by genetic engineering techniques (for general backgroundsee, e.g., Nicholl, in An Introduction to Genetic Engineering, CambridgeUniversity Press: Cambridge (1994), pp. 1-5 & 127-130; Steinberg et al.,in Recombinant DNA Technology Concepts and Biomedical Applications,Prentice Hall: Englewood Cliffs, N.J. (1993), pp. 81-124 & 150-162;Sofer, in Introduction to Genetic Engineering, Butterworth-Heinemann,Stoneham, Mass. (1991), pp. 1-21 & 103-126; Old et al., in Principles ofGene Manipulation, Blackwell Scientific Publishers: London (1992), pp.1-13 & 108-221; and Emtage, in Delivery Systems for Peptide Drugs, Daviset al., eds., Plenum Press: New York (1986), pp. 23-33). Subsequently,the recombinantly produced protein can be isolated and purified usingstandard techniques known in the art (e.g., chromatography,centrifugation, differential solubility, electrophoretic techniques,etc.), and assayed for antiviral activity.

Alternatively, a wild-type scytovirin can be obtained from Scytonemavarium by non-recombinant methods (e.g., see Example 1 and above), andsequenced by conventional techniques. The sequence can then be used todesign and synthesize the corresponding DNA, which can be subcloned intoan appropriate expression vector and delivered into a protein-producingcell for en mass recombinant production of the desired protein.

In view of the above, the present invention provides a method ofinhibiting a viral infection of a host. The method comprisesadministering a viral infection-inhibiting amount of at least one of thefollowing:

-   -   (i) an isolated or purified antiviral protein consisting        essentially of the amino acid sequence of SEQ ID NO: 1, an amino        acid sequence that is about 90% or more identical to SEQ ID NO:        1, an amino acid sequence that is about 90% or more homologous        to SEQ ID NO: 1, or an antiviral fragment of any of the        foregoing,    -   (ii) a variant of (i), which comprises (a) one or more        conservative or neutral amino acid substitutions and/or (b) 1, 2        or 3 amino acid additions at the N-terminus and/or C-terminus,        with the proviso that the variant has antiviral activity        characteristic of the antiviral protein consisting essentially        of the amino acid sequence of SEQ ID NO: 1 and isolated or        purified from Scytonema varium to a greater or lesser extent but        not negated,    -   (iii) a fusion protein of (i),    -   (iv) a fusion protein of (ii),    -   (v) a conjugate comprising (i) and at least one effector        component,    -   (vi) a conjugate comprising (ii) and at least one effector        component,    -   (vii) a composition comprising one or more of (i)-(vi),    -   (viii) an isolated or purified nucleic acid consisting        essentially of a nucleotide sequence encoding the amino acid        sequence of SEQ ID NO: 1, an amino acid sequence that is about        90% or more identical to SEQ ID NO: 1, an amino acid sequence        that is about 90% or more homologous to SEQ ID NO: 1, or an        antiviral fragment of any of the foregoing, optionally in the        form of a vector,    -   (ix) a variant of (viii), which comprises nucleotides        encoding (a) one or more conservative or neutral amino acid        substitutions and/or (b) up to 1, 2 or 3 amino acid additions at        the N-terminus and/or C-terminus, with the proviso that the        encoded amino acid sequence has antiviral activity        characteristic of the antiviral protein, which consists        essentially of the amino acid sequence of SEQ ID NO: 1 and which        is isolated or purified from Scytonema varium, optionally in the        form of a vector,    -   (x) an isolated or purified nucleic acid consisting essentially        of a nucleotide sequence encoding a fusion protein of (viii),        optionally in the form of a vector,    -   (xi) an isolated or purified nucleic acid consisting essentially        of a nucleotide sequence encoding a fusion protein of (ix),        optionally in the form of a vector,    -   (xii) a composition comprising one or more of (viii)-(xi), and    -   (xiii) an isolated cell comprising (viii), (ix), (x), or (xi).        By “viral infection-inhibiting amount” is meant an amount of the        active agent sufficient to inhibit viral infection. The dose        administered to a host, such as an animal, in particular a        human, in the context of the present invention should be        sufficient to effect a prophylactic or therapeutic response in        the individual over a reasonable time frame. The dose used to        achieve a desired antiviral concentration in vivo (e.g.,        0.1-1,000 nM) will be determined by the potency of the        particular active agent employed, the severity of the disease        state of the infected individual, as well as, in the case of        systemic administration, the body weight and age of the infected        individual. The size of the dose also will be determined by the        existence of any adverse side effects that may accompany the        particular active agent employed. It is always desirable,        whenever possible, to keep adverse side effects to a minimum.        The dosages of ddC and AZT used in AIDS or ARC patients have        been published. A virustatic range of ddC is generally between        0.05 μM to 1.0 μM. A range of about 0.005-0.25 mg/kg body weight        is virustatic in most patients. The preliminary dose ranges for        oral administration are somewhat broader, for example 0.001 to        0.25 mg/kg given in one or more doses at intervals of 2, 4, 6,        8, 12, etc. hours. Currently, 0.01 mg/kg body weight ddC given        every 8 hrs is preferred. When given in combined therapy, the        other antiviral agent, for example, can be given at the same        time as the present inventive active agent or the dosing can be        staggered as desired. The two drugs also can be combined in a        composition. Doses of each can be less when used in combination        than when either is used alone.

In terms of administration of the present inventive antiviral agents orconjugates thereof, the dosage can be in unit dosage form, such as atablet or capsule. The term “unit dosage form” as used herein refers tophysically discrete units suitable as unitary dosages for human andanimal subjects, each unit containing a predetermined quantity of ascytovirin, or antiviral fragment, fusion protein or conjugate thereof,alone or in combination with other active agents, calculated in anamount sufficient to produce the desired effect in association with apharmaceutically acceptable diluent, carrier, or vehicle.

The specifications for the unit dosage forms of the present inventiondepend on the particular scytovirin, or antiviral fragment, fusionprotein or conjugate thereof, employed and the effect to be achieved, aswell as the associated pharmacodynamics in the host. The doseadministered should be an “antiviral effective amount” or an amountnecessary to achieve an “effective level” in the individual patient.

Since the “effective level” is used as the preferred endpoint fordosing, the actual dose and schedule can vary, depending uponinterindividual differences in pharmacokinetics, drug distribution, andmetabolism. The “effective level” can be defined, for example, as theblood or tissue level (e.g., 0.1-1,000 nM) desired in the patient thatcorresponds to a concentration of one or more active agents, whichinhibits a virus, such as HIV, in an assay known to predict for clinicalantiviral activity of chemical compounds and biological agents. The“effective level” for agents of the present invention also can vary whenthe present inventive active agent is used in combination with otherknown active agents or combinations thereof.

One skilled in the art can easily determine the appropriate dose,schedule, and method of administration for the exact formulation of thecomposition being used, in order to achieve the desired “effectiveconcentration” in the individual patient. One skilled in the art alsocan readily determine and use an appropriate indicator of the “effectiveconcentration” of the compounds of the present invention by a direct(e.g., analytical chemical analysis) or indirect (e.g., with surrogateindicators such as p24 or RT) analysis of appropriate patient samples(e.g., blood and/or tissues).

In the treatment of some virally infected individuals, it can bedesirable to utilize a “mega-dosing” regimen, wherein a large dose ofthe scytovirin, or antiviral fragment, fusion protein or conjugatethereof, is administered, time is allowed for the drug to act, and thena suitable reagent, device or procedure is administered to theindividual to inactivate or remove the drug.

The method can be used to inhibit viral infection in a hosttherapeutically or prophylactically. By “therapeutically” is meant thatthe host already has been infected with the virus. By “prophylactically”is meant that the host has not yet been infected with the virus but isat risk of being infected with the virus. Prophylactic treatment isintended to encompass any degree of inhibition of viral infection,including, but not limited to, complete inhibition, as one of ordinaryskill in the art will readily appreciate that any degree in inhibitionof viral infection is advantageous. Preferably, the present inventiveactive agent is administered before viral infection or immediately upondetermination of viral infection and is continuously administered untilthe virus is undetectable. The method optionally further comprises theprior, simultaneous or subsequent administration, by the same route or adifferent route, of an antiviral agent or another agent that isefficacious in inhibiting the viral infection. Preferably, the infectionis caused by a virus having as a coat protein a glycoprotein comprisinga high-mannose oligosaccharide, such as an immunodeficiency virus, inwhich case the host is preferably a human and the immunodeficiency virusis preferably human immunodeficiency virus (HIV).

In one embodiment of the method, the isolated cell is a cell from thehost, which had been previously isolated and contacted with (viii),(ix), (x) or (xi). In another embodiment of the method, the isolatedcell is a cell from a homologous host. In yet another embodiment of themethod, the isolated cell is a nonpathogenic bacterium or a yeast.Preferably, the nonpathogenic bacterium is a lactobacillus. Theinsertion of a DNA sequence of a scytovirin (or antiviral fragmentthereof) or fusion protein or conjugate thereof of the present inventionex vivo into cells previously removed from a given animal, such as amammal, in particular a human, host is within the ordinary skill in theart. Such cells express the corresponding scytovirin or fusion proteinor conjugate in vivo after reintroduction into the host. The feasibilityof such a therapeutic strategy to deliver a therapeutic amount of anagent in close proximity to the desired target cells and pathogens,i.e., virus, more particularly retrovirus, specifically HIV and itsenvelope glycoprotein gp120, has been demonstrated in studies with cellsengineered ex vivo to express sCD4 (Morgan et al. (1994), supra). It isalso possible that, as an alternative to ex vivo insertion of the DNAsequences of the present invention, such sequences can be inserted intocells directly in vivo, such as by use of an appropriate viral vector.Such cells transfected in vivo are expected to produce antiviral amountsof a scytovirin or fusion protein or conjugate thereof directly in vivo.

Alternatively, a DNA sequence corresponding to a scytovirin or fusionprotein or conjugate thereof can be inserted into suitable nonmammalianhost cells, and such host cells will express therapeutic or prophylacticamounts of a scytovirin or fusion protein or conjugate thereof directlyin vivo within or onto a desired body compartment of an animal, inparticular a human. In a preferred embodiment of the present invention,a method of female-controllable prophylaxis against viral infection,such as HIV infection, comprises the intravaginal administration and/orestablishment of, in a female human, a persistent intravaginalpopulation of lactobacilli that have been transformed with a codingsequence of the present invention to produce, over a prolonged time,effective virucidal levels of a scytovirin or fusion protein orconjugate thereof, directly on or within or onto the vaginal and/orcervical and/or uterine mucosa. It is noteworthy that both of the WorldHealth Organization (WHO), as well as the U.S. National Institute ofAllergy and Infectious Diseases, have pointed to the need fordevelopment of female-controlled topical microbicides, suitable forblocking the transmission of HIV, as an urgent global priority (Lange etal., Lancet 341: 1356 (1993); and Fauci, NIAID News, Apr. 27, 1995).

Scytovirins and fusion proteins and conjugates thereof collectivelycomprise proteins and peptides, and, as such, are particularlysusceptible to hydrolysis of amide bonds (e.g., catalyzed by peptidases)and disruption of essential disulfide bonds or formation of inactivatingor unwanted disulfide linkages (Carone et al., J. Lab. Clin. Med. 100:1-14 (1982)). There are various ways to alter molecular structure, ifnecessary, to provide enhanced stability to the scytovirin or conjugatethereof (Wunsch, Biopolymers 22: 493-505 (1983); and Samanen, inPolymeric Materials in Medication, Gebelein et al., eds., Plenum Press:New York (1985), pp. 227-242), which may be essential for preparationand use of pharmaceutical compositions containing scytovirins orconjugates thereof for therapeutic or prophylactic applications againstviruses, e.g., HIV. Possible options for useful chemical modificationsof a scytovirin or fusion protein or conjugate thereof include, but arenot limited to, the following (adapted from Samanen (1985), supra): (a)olefin substitution, (b) carbonyl reduction, (c) D-amino acidsubstitution, (d) N-methyl substitution, (e) C-methyl substitution, (f)C-C′-methylene insertion, (g) dehydro amino acid insertion, (h)retro-inverso modification, (I) N-terminal to C-terminal cyclization,and (j) thiomethylene modification. Scytovirins and fusion proteins andconjugates thereof also can be modified by covalent attachment ofcarbohydrate and polyoxyethylene derivatives, which are expected toenhance stability and resistance to proteolysis (Abuchowski et al., inEnzymes as Drugs, Holcenberg et al., eds., John Wiley: New York (1981),pp. 367-378).

Other important general considerations for design of delivery strategysystems and compositions, and for routes of administration, for proteinand peptide drugs, such as scytovirins and fusion proteins andconjugates thereof (Eppstein, CRC Crit. Rev. Therapeutic Drug CarrierSystems 5: 99-139 (1988); Siddiqui et al., CRC Crit. Rev. TherapeuticDrug Carrier Systems 3: 195-208 (1987); Banga et al., Int. J.Pharmaceutics 48: 15-50 (1988); Sanders, Eur. J. Drug Metab.Pharmacokinetics 15: 95-102 (1990); and Verhoef, Eur. J. Drug Metab.Pharmacokinetics 15: 83-93 (1990), also apply. The appropriate deliverysystem for a given scytovirin or fusion protein or conjugate thereofwill depend upon its particular nature, the particular clinicalapplication, and the site of drug action. As with any protein or peptidedrug, oral delivery of a scytovirin or a conjugate thereof will likelypresent special problems, due primarily to instability in thegastrointestinal tract and poor absorption and bioavailability ofintact, bioactive drug therefrom. Therefore, especially in the case oforal delivery, but also possibly in conjunction with other routes ofdelivery, it may be desirable to use an absorption-enhancing agent incombination with a given scytovirin or fusion protein or conjugatethereof. A wide variety of absorption-enhancing agents have beeninvestigated and/or applied in combination with protein and peptidedrugs for oral delivery and for delivery by other routes (Verhoef, 1990,supra; van Hoogdalem, Pharmac. Ther. 44: 407-443 (1989); Davis, J.Pharm. Pharmacol. 44(Suppl. 1): 186-190 (1992)). Most commonly, typicalenhancers fall into the general categories of (a) chelators, such asEDTA, salicylates, and N-acyl derivatives of collagen, (b) surfactants,such as lauryl sulfate and polyoxyethylene-9-lauryl ether, (c) bilesalts, such as glycholate and taurocholate, and derivatives, such astauro-di-hydro-fusidate, (d) fatty acids, such as oleic acid and capricacid, and their derivatives, such as acylcarnitines, monoglycerides anddiglycerides, (e) non-surfactants, such as unsaturated cyclic ureas, (f)saponins, (g) cyclodextrins, and (h) phospholipids.

Other approaches to enhancing oral delivery of protein and peptidedrugs, such as the scytovirins and fusion proteins and conjugatesthereof, can include aforementioned chemical modifications to enhancestability to gastrointestinal enzymes and/or increased lipophilicity.Alternatively, or in addition, the protein or peptide drug can beadministered in combination with other drugs or substances, whichdirectly inhibit proteases and/or other potential sources of enzymaticdegradation of proteins and peptides. Yet another alternative approachto prevent or delay gastrointestinal absorption of protein or peptidedrugs, such as scytovirins or fusion proteins or conjugates thereof, isto incorporate them into a delivery system that is designed to protectthe protein or peptide from contact with the proteolytic enzymes in theintestinal lumen and to release the intact protein or peptide only uponreaching an area favorable for its absorption. A more specific exampleof this strategy is the use of biodegradable microcapsules ormicrospheres, both to protect vulnerable drugs from degradation, as wellas to effect a prolonged release of active drug (Deasy, inMicroencapsulation and Related Processes, Swarbrick, ed., MarcellDekker, Inc.: New York (1984), pp. 1-60, 88-89, 208-211). Microcapsulesalso can provide a useful way to effect a prolonged delivery of aprotein and peptide drug, such as a scytovirin or conjugate thereof,after injection (Maulding, J. Controlled Release 6: 167-176 (1987)).

Given the aforementioned potential complexities of successful oraldelivery of a protein or peptide drug, it is fortunate that there arenumerous other potential routes of delivery of a protein or peptidedrug, such as a scytovirin or fusion protein or conjugate thereof. Theseroutes include intravenous, intraarterial, intrathecal, intracisternal,buccal, rectal, nasal, pulmonary, transdermal, vaginal, ocular, and thelike (Eppstein (1988), supra; Siddiqui et al. (1987), supra; Banga etal. (1988), supra; Sanders (1990), supra; Verhoef (1990), supra; Barry,in Delivery Systems for Peptide Drugs, Davis et al., eds., Plenum Press:New York (1986), pp. 265-275; and Patton et al., Adv. Drug Delivery Rev.8: 179-196 (1992)). With any of these routes, or, indeed, with any otherroute of administration or application, a protein or peptide drug, suchas a scytovirin or fusion protein or conjugate thereof, may initiate animmunogenic reaction. In such situations it may be necessary to modifythe molecule in order to mask immunogenic groups. It also can bepossible to protect against undesired immune responses by judiciouschoice of method of formulation and/or administration. For example,site-specific delivery can be employed, as well as masking ofrecognition sites from the immune system by use or attachment of aso-called tolerogen, such as polyethylene glycol, dextran, albumin, andthe like (Abuchowski et al. (1981), supra; Abuchowski et al., J. Biol.Chem. 252: 3578-3581 (1977); Lisi et al., J. Appl. Biochem. 4: 19-33(1982); and Wileman et al., J. Pharm. Pharmacol. 38: 264-271 (1986)).Such modifications also can have advantageous effects on stability andhalf-life both in vivo and ex vivo. Procedures for covalent attachmentof molecules, such as polyethylene glycol, dextran, albumin and thelike, to proteins, such as scytovirins or fusion proteins or conjugatesthereof, are well-known to those skilled in the art, and are extensivelydocumented in the literature (e.g., see Davis et al., In Peptide andProtein Drug Delivery, Lee, ed., Marcel Dekker: New York (1991), pp.831-864).

Other strategies to avoid untoward immune reactions can also include theinduction of tolerance by administration initially of only low doses. Inany event, it will be apparent from the present disclosure to oneskilled in the art that, for any particular desired medical applicationor use of a scytovirin or fusion protein or conjugate thereof, theskilled artisan can select from any of a wide variety of possiblecompositions, routes of administration, or sites of application, what isadvantageous.

The present inventive compositions can be used in the context of thepresent inventive method in combination with other active agents toinhibit viral infection as a result of sexual transmission. Potentialagents used or being considered for use against sexual transmission ofHIV are very limited; present agents in this category include, forexample, nonoxynol-9 (Bird, AIDS 5: 791-796 (1991)), gossypol andderivatives (Polsky et al., Contraception 39: 579-587 (1989); Lin,Antimicrob. Agents Chemother. 33: 2149-2151 (1989); and Royer,Pharmacol. Res. 24: 407-412 (1991)), and gramicidin (Bourinbair, LifeSci./Pharmacol. Lett. 54: PL5-9 (1994); and Bourinbair et al.,Contraception 49: 131-137 (1994)). The method of prevention of sexualtransmission of viral infection, e.g., HIV infection, in accordance withthe present invention comprises vaginal, rectal, oral, penile or othertopical treatment with a viral-infection inhibiting amount of ascytovirin and/or scytovirin fusion protein and/or scytovirin conjugate,alone or in combination with another antiviral agent as described above.

Nonpathogenic commensal bacteria and yeasts also offer an attractivemeans of in situ delivery of scytovirins or antiviral derivativesthereof to prevent sexual transmission of viral infections. For example,lactobacilli readily populate the vagina, and indeed are a predominantbacterial population in most healthy women (Redondo-Lopez et al., Rev.Infect. Dis. 12: 856-872 (1990); Reid et al., Clin. Microbiol. Rev. 3:335-344 (1990); Bruce and Reid, Can. J. Microbiol. 34: 339-343 (1988);Reu et al., J. Infect. Dis. 171: 1237-1243 (1995); Hilier et al., Clin.Infect. Dis. 16(Suppl 4): S273-S281; and Agnew et al., Sex. Transm. Dis.22: 269-273 (1995)). Lactobacilli are also prominent, nonpathogenicinhabitants of other body cavities, such as the mouth, nasopharynx,upper and lower gastrointestinal tracts, and rectum.

It is well-established that lactobacilli can be readily transformedusing available genetic engineering techniques to incorporate a desiredforeign DNA sequence, and that such lactobacilli can be made to expressa corresponding desired foreign protein (see, e.g., Hols et al., Appl.and Environ. Microbiol. 60: 1401-1413 (1994)). Therefore, within thecontext of the present disclosure, it will be appreciated by one skilledin the art that viable host cells containing a DNA sequence or vector ofthe present invention, and expressing a protein or peptide of thepresent invention, can be used directly as the delivery vehicle for ascytovirin or fusion protein or conjugate thereof to the desired site(s)in vivo. Preferred host cells for such delivery of scytovirins orconjugates thereof directly to desired site(s), such as, for example, toa selected body cavity, can comprise bacteria. More specifically, suchhost cells can comprise suitably engineered strain(s) of lactobacilli,enterococci, or other common bacteria, such as E. coli, normal strainsof which are known to commonly populate body cavities. More specificallyyet, such host cells can comprise one or more selected nonpathogenicstrains of lactobacilli, such as those described by Andreu et al. (1995,supra), especially those having high adherence properties to epithelialcells, such as, for example, adherence to vaginal epithelial cells, andsuitably transformed using the DNA sequences of the present invention.

As reviewed by McGroarty (FEMS Immunol. Med. Microbiol. 6: 251-264(1993)) the “probiotic” or direct therapeutic application of livebacteria, particularly bacteria that occur normally in nature, moreparticularly lactobacilli, for treatment or prophylaxis againstpathogenic bacterial or yeast infections of the urogenital tract, inparticular the female urogenital tract, is a well-established concept.However, present inventive use of non-mammalian cells, particularlybacteria, more particularly lactobacilli, specifically engineered with ascytovirin gene, to express a scytovirin, is heretofore unprecedented asa method of treatment of an animal, specifically a human, to preventinfection by a virus, specifically a retrovirus, more specifically HIV-1or HIV-2.

Elmer et al. (JAMA 275: 870-876 (1996)) have recently speculated that“genetic engineering offers the possibility of using microbes to deliverspecific actions or products to the colon or other mucosal surfaces . .. other fertile areas for future study include defining the mechanismsof action of various biotherapeutic agents with the possibility ofapplying genetic engineering to enhance activities.” Elmer et al. (1996,supra) further point out that the terms “probiotic” and “biotherapeuticagent” have been used in the literature to describe microorganisms thathave antagonistic activity toward pathogens in vivo; those authors morespecifically prefer the term “biotherapeutic agent” to denote“microorganisms having specific therapeutic properties.”

In view of the present disclosure, one skilled in the art willappreciate that the present invention teaches an entirely novel type of“probiotic” or “biotherapeutic” treatment using specifically engineeredstrains of microorganisms provided herein which do not occur in nature.Nonetheless, available teachings concerning selection of optimalmicrobial strains, in particular bacterial strains, for conventionalprobiotic or biotherapeutic applications can be employed in the contextof the present invention. For example, selection of optimallactobacillus strains for genetic engineering, transformation, directexpression of scytovirins or fusion proteins or conjugates thereof, anddirect probiotic or biotherapeutic applications, to treat or preventviral, e.g., HIV, infection, can be based upon the same or similarcriteria, such as those described by Elmer et al. (1996), supra,typically used to select normal, endogenous or “nonengineered” bacterialstrains for conventional probiotic or biotherapeutic therapy.Furthermore, the recommendations and characteristics taught byMcGroarty, particularly for selection of optimal lactobacillus strainsfor conventional probiotic use against female urogenital infections, arepertinent to the present invention: “ . . . lactobacilli chosen forincorporation into probiotic preparations should be easy and, ifpossible, inexpensive to cultivate . . . strains should be stable,retain viability following freeze-drying and, of course, benon-pathogenic to the host . . . it is essential that lactobacillichosen for use in probiotic preparations should adhere well to thevaginal epithelium . . . ideally, artificially implanted lactobacillishould adhere to the vaginal epithelium, integrate with the indigenousmicroorganisms present, and proliferate” (MceGroarty (1993), supra).While McGroarty's teachings specifically address selections of “normal”lactobacillus strains for probiotic uses against pathogenic bacterial oryeast infections of the female urogenital tract, similar considerationswill apply to the selection of optimal bacterial strains for geneticengineering and “probiotic” or “biotherapeutic” application againstviral infections as particularly encompassed by the present invention.

Accordingly, the method of the present invention for the prevention ofsexual transmission of viral infection, e.g., HIV infection, comprisesvaginal, rectal, oral, penile, or other topical, insertional, orinstillational treatment with a viral infection-inhibiting amount of ascytovirin or fusion protein or conjugate thereof, and/or viable hostcells transformed to express a scytovirin or conjugate thereof, alone orin combination with one or more other antiviral agents (e.g., asdescribed above).

One skilled in the art will appreciate that various routes ofadministering a drug are available, and, although more than one routecan be used to administer a particular drug, a particular route canprovide a more immediate and more effective reaction than another route.Furthermore, one skilled in the art will appreciate that the particularpharmaceutical carrier employed will depend, in part, upon theparticular scytovirin or fusion protein or conjugate thereof employed,and the chosen route of administration. Accordingly, there is a widevariety of suitable formulations of the composition of the presentinvention.

Formulations suitable for oral administration can consist of liquidsolutions, such as an effective amount of the compound dissolved indiluents, such as water, saline, or fruit juice; capsules, sachets ortablets, each containing a predetermined amount of the activeingredient, as solid, granules or freeze-dried cells; solutions orsuspensions in an aqueous liquid; oil-in-water emulsions or water-in-oilemulsions; lozenges comprising the active ingredient in a flavor,usually sucrose and acacia or tragacanth; pastilles comprising theactive ingredient in an inert base, such as gelatin and glycerin, orsucrose and acacia; and mouthwashes comprising the active ingredient ina suitable liquid carrier; as well as creams, emulsions, gels and thelike containing, in addition to the active ingredient, such as, forexample, freeze-dried lactobacilli or live lactobacillus culturesgenetically engineered to directly produce a scytovirin or fusionprotein or conjugate thereof of the present invention, such carriers asare known in the art. Tablet forms can include one or more of lactose,mannitol, corn starch, potato starch, microcrystalline cellulose,acacia, gelatin, colloidal silicon dioxide, croscarmellose sodium, talc,magnesium stearate, stearic acid, and other excipients, colorants,diluents, buffering agents, moistening agents, preservatives, flavoringagents, and pharmacologically compatible carriers. Suitable formulationsfor oral delivery can also be incorporated into synthetic and naturalpolymeric microspheres, or other means to protect the agents of thepresent invention from degradation within the gastrointestinal tract(see, for example, Wallace et al., Science 260: 912-915 (1993)).

The scytovirins or fusion proteins or conjugates thereof, alone or incombination with other antiviral agents, can be made into aerosolformulations or microparticulate powder formulations to be administeredvia inhalation. These aerosol formulations can be placed intopressurized acceptable propellants, such as dichlorodifluoromethane,propane, nitrogen and the like.

The scytovirins or fusion proteins or conjugates thereof, alone or incombinations with other antiviral agents or absorption modulators, canbe made into suitable formulations for transdermal application andabsorption (Wallace et al. (1993), supra). Transdermal electroporationor iontophoresis also can be used to promote and/or control the systemicdelivery of the compounds and/or compositions of the present inventionthrough the skin (e.g., see Theiss et al., Meth. Find. Exp. Clin.Pharmacol. 13: 353-359 (1991)).

Formulations for rectal administration can be presented as a suppositorywith a suitable base comprising, for example, cocoa butter or asalicylate. Formulations suitable for vaginal administration can bepresented as pessaries, tampons, creams, gels, pastes, foams, or sprayformulas containing, in addition to the active ingredient, such as, forexample, freeze-dried lactobacilli or live lactobacillus culturesgenetically engineered to directly produce a scytovirin or fusionprotein or conjugate thereof of the present invention, such carriers asare known in the art to be appropriate. Similarly, the active ingredientcan be combined with a lubricant as a coating on a condom. Indeed,preferably, the active ingredient is applied to and/or delivered by anycontraceptive device, including, but not limited to, a condom, adiaphragm, a cervical cap, a vaginal ring and a sponge.

Formulations suitable for parenteral administration include aqueous andnon-aqueous, isotonic sterile injection solutions, which can containanti-oxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.The formulations can be presented in unit-dose or multi-dose sealedcontainers, such as ampules and vials, and can be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid carrier, for example, water, for injections, immediatelyprior to use. Extemporaneous injection solutions and suspensions can beprepared from sterile powders, granules, and tablets of the kindpreviously described.

The present invention further provides a method of inhibiting a virus ina biological sample or in/on an inanimate object. The method comprisescontacting the biological sample or the inanimate object with aviral-inhibiting amount of at least one of the following:

-   -   (i) an isolated or purified antiviral protein consisting        essentially of the amino acid sequence of SEQ ID NO: 1, an amino        acid sequence that is about 90% or more identical to SEQ ID NO:        1, an amino acid sequence that is about 90% or more homologous        to SEQ ID NO: 1, or an antiviral fragment of any of the        foregoing,    -   (ii) a variant of (i), which comprises (a) one or more        conservative or neutral amino acid substitutions and/or (b) 1, 2        or 3 amino acid additions at the N-terminus and/or C-terminus,        with the proviso that the variant has antiviral activity        characteristic of the antiviral protein, which consists        essentially of the amino acid sequence of SEQ ID NO: 1 and which        is isolated or purified from Scytonema varium, to a greater or        lesser extent but not negated,    -   (iii) a fusion protein of (i),    -   (iv) a fusion protein of (ii),    -   (v) a conjugate comprising (i) and at least one effector        component,    -   (vi) a conjugate comprising (ii) and at least one effector        component, and    -   (vii) a composition comprising one or more of (i)-(vi). By        “viral-inhibiting” amount is meant an amount of active agent,        such as in the range of 0.1-1,000 nM, sufficient to inhibit the        virus so as to reduce, and desirably eliminate, its infectivity.        The method optionally further comprises the prior, simultaneous        or subsequent contacting, in the same manner or a different        manner, of the biological sample or inanimate object with an        antiviral agent or another agent that is efficacious in        inhibiting the virus. The biological sample can be blood, a        blood product, cells, a tissue, an organ, sperm, a vaccine        formulation, a bodily fluid, and the like. When the sample is a        vaccine formulation, preferably the virus that is inhibited is        infectious, such as HIV, although HIV, such as infectious HIV,        can be inhibited in other samples in accordance with this        method. The inanimate object can be a solution, a medical        supply, or a medical equipment. Fusion proteins and effector        components are as described above.

Formulations comprising a scytovirin or fusion protein or conjugatethereof suitable for virucidal (e.g., HIV) sterilization of inanimateobjects, such as medical supplies or equipment, laboratory equipment andsupplies, instruments, devices, and the like, can, for example, beselected or adapted as appropriate, by one skilled in the art, from anyof the aforementioned compositions or formulations. Preferably, thescytovirin is produced by recombinant DNA technology. The scytovirinfusion protein can be produced by recombinant DNA technology, whereasthe conjugate can be produced by chemical coupling of a scytovirin withan effector component as described above. Similarly, formulationssuitable for ex vivo sterilization, or inhibition of virus, such asinfectious virus, in a sample, such as blood, a blood product, sperm, orother bodily products, such as a fluid, cells, a tissue or an organ, orany other solution, suspension, emulsion, vaccine formulation or othermaterial which can be administered to a patient in a medical procedure,can be selected or adapted as appropriate by one skilled in the art,from any of the aforementioned compositions or formulations. However,suitable formulations for ex vivo sterilization or inhibition of virusfrom a sample or in/on an inanimate object are by no means limited toany of the aforementioned formulations or compositions. For example,such formulations or compositions can comprise a functional scytovirin,such as that which is encoded by SEQ ID NO:1, or antiviral fragment,fusion protein or conjugate thereof, attached to a solid support matrix,to facilitate contacting, or otherwise inhibiting infectious virus in asample such as described above, e.g., a bodily product such as a fluid,cells, a tissue or an organ from an organism, in particular a mammal,such as a human, including, for example, blood, a component of blood, orsperm. Preferably, the antiviral protein consists essentially of SEQ IDNO:1. Also preferably, the protein binds gp120 of HIV, in particularinfectious HIV. As a more specific example, such a formulation orcomposition can comprise a functional scytovirin, or fusion protein orconjugate thereof, attached to (e.g., coupled to or immobilized on) asolid support matrix comprising magnetic beads, to facilitate contactingand inhibition of infectious virus, and enabling magnet-assisted removalof the bead-bound scytovirin or conjugate thereof from a sample asdescribed above, e.g., a bodily product such as a fluid, cells, a tissueor an organ, blood, a component of blood, or sperm. Alternatively, andalso preferably, the solid support matrix comprises a contraceptivedevice, such as a condom, a diaphragm, a cervical cap, a vaginal ring ora sponge.

As an even more specific illustration, such a composition (e.g., for exvivo use) can comprise a functional scytovirin, or antiviral fragment,fusion protein or conjugate thereof, attached to a solid support matrix,such as magnetic beads or a flow-through matrix, by means of ananti-scytovirin antibody or at least one effector component, which canbe the same or different, such as polyethylene glycol, albumin ordextran. The conjugate can further comprise at least one effectorcomponent, which can be the same or different, selected from the groupconsisting of an immunological reagent, a toxin and an antiviral agent.A flow-through matrix would comprise, for instance, a configurationsimilar to an affinity column. The scytovirin can be covalently coupledto a solid support matrix via an anti-scytovirin antibody, describedbelow. Methods of attaching an antibody to a solid support matrix arewell-known in the art (see, for example, Harlow and Lane. Antibodies: ALaboratory Manual, Cold Springs Harbor Laboratory: Cold Spring Harbor,N.Y., 1988). Alternatively, the solid support matrix, such as magneticbeads, can be coated with streptavidin, in which case the scytovirin, ora fusion protein or a conjugate thereof, is biotinylated. Such acomposition can be prepared, for example, by biotinylating thescytovirin, or antiviral fragment, fusion protein or conjugate thereof,and then contacting the biotinylated scytovirin with a (commerciallyavailable) solid support matrix, such as magnetic beads, coated withstreptavidin. The use of biotinylation as a means to attach a desiredbiologically active protein to a streptavidin-coated support matrix,such as magnetic beads, is well-known in the art. One skilled in the artwill appreciate that a suitable or appropriate formulation can beselected, adapted or developed based upon the particular application athand.

Other types of means, as are known in the art, can be used to attach afunctional scytovirin (or an antiviral fragment, fusion protein orconjugate thereof as described above) to a solid support matrix, such asa magnetic bead, in which case contact with a magnet is used to separatethe sample and the composition. For instance, the skilled practitionermight select a poly(ethylene glycol) molecule for attaching a functionalscytovirin to a solid support matrix, thereby to provide amatrix-anchored scytovirin, wherein the scytovirin is attached to thematrix by a longer “tether” than would be feasible or possible for otherattachment methods, such as biotinylation/streptavidin coupling. Ascytovirin coupled by a poly(ethylene glycol) “tether” to a solidsupport matrix (such as magnetic beads, porous surface or membrane, andthe like) can permit optimal exposure of a binding surface, epitope,hydrophobic or hydrophilic focus, and/or the like, on a functionalscytovirin in a manner that, in a given situation and/or for aparticular virus, facilitates inhibition of the virus.

Similarly, other types of solid support matrices can be used, such as amatrix comprising a porous surface or membrane, over or through which asample is flowed or percolated, thereby selectively inhibitinginfectious virus in the sample. The choice of solid support matrix,means of attachment of the functional scytovirin to the solid supportmatrix, and means of separating the sample and the matrix-anchoredscytovirin will depend, in part, on the sample (e.g., fluid vs. tissue)and the virus to be inhibited. It is expected that the use of a selectedcoupling molecule can confer certain desired properties to a matrix,comprising a functional scytovirin coupled therewith, that may haveparticularly advantageous properties in a given situation.

Such methods also have utility in real time ex vivo inhibition of virusor virus infected cells in a bodily fluid, such as blood, e.g., in thetreatment of viral infection, or in the inhibition of virus in blood ora component of blood, e.g., for transfusion, in the inhibition orprevention of viral infection. Such methods also have potential utilityin dialysis, such as kidney dialysis, and in inhibiting virus in spermobtained from a donor for in vitro and in vivo fertilization. Themethods also have applicability in the context of tissue and organtransplantations.

The present invention also provides antibodies directed to the proteinsof the present invention. The availability of antibodies to any givenprotein is highly advantageous, as it provides the basis for a widevariety of qualitative and quantitative analytical methods, separationand purification methods, and other useful applications directed to thesubject proteins. Accordingly, given the present disclosure and theproteins of the present invention, it will be readily apparent to oneskilled in the art that antibodies, in particular antibodiesspecifically binding to a protein of the present invention, can beprepared using well-established methodologies (e.g., such as themethodologies described in detail by Harlow and Lane, in Antibodies. ALaboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor,1988, pp. 1-725). Such antibodies can comprise both polyclonal andmonoclonal antibodies. Furthermore, such antibodies can be obtained andemployed either in solution-phase or coupled to a desired solid-phasematrix, such as magnetic beads or a flow through matrix. Having in handsuch antibodies as provided by the present invention, one skilled in theart will further appreciate that such antibodies, in conjunction withwell-established procedures (e.g., such as described by Harlow and Lane(1988, supra)) comprise useful methods for the detection,quantification, or purification of a scytovirin, or antiviral fragment,fusion protein or conjugate thereof, or host cell transformed to producethe same. Example 2 further illustrates an antibody specifically bindinga scytovirin. Preferably, the antibody binds to an epitope of scytovirinconsisting essentially of SEQ ID NO: 1, particularly a scytovirin, whichconsists essentially of SEQ ID NO: 1 and which has been purified orisolated from Scytonema varium. In this regard, the present inventionalso provides a composition comprising such an antibody.

Also provided is an anti-scytovirin antibody. Preferably, theanti-scytovirin antibody has an internal image of gp120 of animmunodeficiency virus. In this regard, the present invention furtherprovides a composition comprising such an antibody. The composition canfurther comprise an immunostimulant.

Matrix-anchored anti-scytovirin antibodies can be used in a method toinhibit virus in a sample. Preferably, the antibody binds to an epitopeof a scytovirin consisting essentially of SEQ ID NO: 1. The antibody canbe coupled to a solid support matrix using similar methods and withsimilar considerations as described above for attaching a scytovirin toa solid support matrix. For example, coupling methods and moleculesemployed to attach an anti-scytovirin antibody to a solid supportmatrix, such as magnetic beads or a flow-through matrix, can employbiotin/streptavidin coupling or coupling through molecules, such aspolyethylene glycol, albumin or dextran. Also analogously, it can beshown that, after such coupling, the matrix-anchored anti-scytovirinantibody retains its ability to bind to a scytovirin consistingessentially of SEQ ID NO: 1, which protein can inhibit a virus.Preferably, the matrix is a solid support matrix, such as a magneticbead or a flow-through matrix. If the solid support matrix to which theanti-scytovirin antibody is attached comprises magnetic beads, removalof the antibody-scytovirin complex can be readily accomplished using amagnet.

The present invention also provides an anti-scytovirin antibody that isanti-idiotypic in respect to gp120, i.e., has an internal image of gp120of a primate immunodeficiency virus. Preferably, the antibody cancompete with gp120 of a primate immunodeficiency virus for binding to ascytovirin. In this regard, the primary immunodeficiency viruspreferably is HIV-1 or HIV-2 and the scytovirin preferably consistsessentially of SEQ ID NO:1. Anti-idiotypic antibodies can be generatedin accordance with methods known in the art (see, for example, Benjamin,In Immunology: a short course, Wiley-Liss, NY, 1996, pp. 436-437; Kuby,In Immunology, 3rd ed., Freeman, NY, 1997, pp. 455-456; Greenspan, etal., FASEB J. 7: 437-443 (1993); and Poskitt, Vaccine 9: 792-796(1991)). Such an anti-idiotypic (in respect to gp120) anti-scytovirinantibody is useful in a method of inhibiting infection of an animal witha virus as provided herein.

In view of the above, a scytovirin can be administered to an animal, theanimal generates anti-scytovirin antibodies, among which are antibodiesthat have an internal image of gp120. In accordance with well-knownmethods, polyclonal or monoclonal antibodies can be obtained, isolatedand selected. Selection of an anti-scytovirin antibody that has aninternal image of gp120 can be based upon competition between theanti-scytovirin antibody and gp120 for binding to a scytovirin, or uponthe ability of the anti-scytovirin antibody to bind to a free scytovirinas opposed to a scytovirin bound to gp120. Such an anti-scytovirinantibody can be administered to an animal to inhibit a viral infectionin accordance with methods provided herein. Although nonhumananti-idiotypic antibodies, such as an anti-scytovirin antibody that hasan internal image of gp120 and, therefore, is anti-idiotypic to gp120,are proving useful as vaccine antigens in humans, their favorableproperties might, in certain instances, be further enhanced and/or theiradverse properties further diminished, through “humanization”strategies, such as those recently reviewed by Vaughan, (Nature Biotech.16: 535-539 (1998)). Alternatively, a scytovirin can be directlyadministered to an animal to inhibit a viral infection in accordancewith methods provided herein such that the treated animal, itself,generates an anti-scytovirin antibody that has an internal image ofgp120. The production of anti-idiotypic antibodies, such asanti-scytovirin antibody that has an internal image of gp120 and,therefore, is anti-idiotypic to gp120, in an animal to be treated isknown as “anti-idiotype induction therapy,” and is described byMadiyalakan et al. (Hybridoma 14: 199-203 (1995)), for example.

In view of the above, the present invention enables another method ofinhibiting infection of an animal, such as a mammal, in particular ahuman, with a virus. The method comprises administering to the animal ananti-scytovirin antibody, or a composition comprising same, in an amountsufficient to induce in the animal an immune response to the virus,whereupon the infection of the animal with the virus is inhibited.Preferably, the anti-scytovirin antibody has an internal image of gp120of an immunodeficiency virus with which the animal can be infected, suchas a primate immunodeficiency virus. Preferably, the antibody cancompete with gp120 of a primate immunodeficiency virus for binding to ascytovirin. In this regard, the primate immunodeficiency viruspreferably is HIV-1 or HIV-2 and the scytovirin preferably consistsessentially of SEQ ID NO:1. The method can further comprise theadministration of an immunostimulant.

Also enabled by the present invention is yet another method ofinhibiting infection of an animal, such as a mammal, in particular ahuman, with a virus. The method comprises administering to the animal ascytovirin, which binds gp120 of an immunodeficiency virus with whichthe animal can be infected, in an amount sufficient to induce in theanimal an anti-scytovirin antibody in an amount sufficient to induce animmune response to a virus sufficient to inhibit infection of the animalwith the virus. Preferably, the anti-scytovirin antibody has an internalimage of gp120 of an immunodeficiency virus with which the animal can beinfected, such as a primate immunodeficiency virus. Preferably, theantibody can compete with gp120 of a primate immunodeficiency virus forbinding to a scytovirin. In this regard, the primate immunodeficiencyvirus preferably is HIV-1 or HIV-2 and the scytovirin preferablyconsists essentially of SEQ ID NO:1.

With respect to the above methods, sufficient amounts can be determinedin accordance with methods known in the art. Similarly, the sufficiencyof an immune response in the inhibition of a viral infection in ananimal also can be assessed in accordance with methods known in the art.

Either one of the above methods can further comprise concurrent, pre- orpost-treatment with an adjuvant to enhance the immune response, such asthe prior, simultaneous or subsequent administration, by the same or adifferent route, of an antiviral agent or another agent that isefficacious in inducing an immune response to the virus, such as animmunostimulant. See, for example, Harlow et al., 1988, supra.

The present inventive scytovirins are further described in the contextof the following examples. These examples serve to illustrate furtherthe present invention and are not intended to limit the scope of theinvention.

EXAMPLES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference:

-   Birren et al., Genome Analysis: A Laboratory Manual Series, Volume    1, Analyzing DNA, Cold Spring Harbor Laboratory Press, Cold Spring    Harbor, N.Y. (1997),-   Birren et al., Genome Analysis: A Laboratory Manual Series, Volume    2, Detecting Genes, Cold Spring Harbor Laboratory Press, Cold Spring    Harbor, N.Y. (1998),-   Birren et al., Genome Analysis: A Laboratory Manual Series, Volume    3, Cloning Systems, Cold Spring Harbor Laboratory Press, Cold Spring    Harbor, N.Y. (1999),-   Birren et al., Genome Analysis: A Laboratory Manual Series, Volume    4, Mapping Genomes, Cold Spring Harbor Laboratory Press, Cold Spring    Harbor, N.Y. (1999),-   Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor    Laboratory Press, Cold Spring Harbor, N.Y. (1988),-   Harlow et al., Using Antibodies: A Laboratory Manual, Cold Spring    Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1999), and-   Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd    edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,    N.Y. (1989).

Example 1

This example shows details of anti-HIV bioassay-guided isolation,purification and sequence elucidation of scytovirin from aqueousextracts of the cultured cyanobacterium Scytonema varium.

Experimental details pertinent to Example 1 as well as the subsequentExamples are as follows. All solvents were HPLC grade purchased from EMScience (Gibbstown, N.J.). Endoproteinases Arg-C and Glu-C were obtainedfrom Roche Molecular Biochemicals (Indianapolis, Ind.). The monomericsugars, wheat germ agglutinin, HSA, BSA, aprotinin, bovine IgG, α-acidglycoprotein and Sephadex G-100 were purchased from Sigma Corp. (St.Louis, Mo.). Oligosaccharides were purchased from Glyko, Inc. (Novato,Calif.). The rgp120 (recombinant, glycosylated, HIV-1IIIB gp120), rgp160(recombinant, HIV-1IIIB gp160), and rgp41 (recombinant, HIV-1HxB2 gp41,ecto domain) were obtained from Advanced Biotechnologies Incorporated(Columbia, Md.). The sCD4, glycosylated and nonglycosylated gp120(HIV-1_(SF2) gp120), HIV-1 M-tropic (Ba-L) and T-tropic (IIIB) isolateswere obtained from the National Institute of Allergy and InfectiousDiseases AIDS Research and Reference Program, National Institutes ofHealth (NIH). Origins of the CEM-SS human lymphoblastoid cells and theviral strain HIV_(RF) have been previously described (Gulakowski et al.,J. Virol. Methods 33: 87-100 (1991)).

All HPLC separations were obtained using a Rainin SD-1 system with aKnauer variable wavelength detector monitored at 210 nm and a RaininDynanax C₁₈ 300 Å column (1×25 cm), unless otherwise stated.Electrospray ionization mass spectra were recorded on a Hewlett-PackardHP 1100 integrated LC-MS (liquid chromatograph-mass spectrometer) systemequipped with an electrospray interface. Samples were introduced intothe mass spectrometer at a flow rate of 0.2 mL/min with instrumentalconditions as follows: nebulizer pressure (N₂) 25 psig; drying gas flow(N₂) 10 L/min; drying gas temperature, 350° C.; capillary voltage,4,000; fragmentor voltage, 80; mass range, 250-1,600 amu.

SDS-PAGE was performed by methods previously described (Laemmli, Nature227: 680-685 (1970)) on a Novex apparatus using a 14% polyacrylamideresolving gel (precast, Novex). Gels were run at a constant current of25 mA/gel for 60 min at room temperature. Amino acid sequences weredetermined by sequential Edman degradation using an Applied BiosystemsModel 494 sequencer according to the protocols of the manufacturer. TheGenbank nonredundant database, BLASTP, was used to search for N-terminalamino acid sequence similarity as described (Altschul et al., NucleicAcid Res. 25: 3389-3402 (1997)).

The method described in Weislow et al. ((1989), supra) was used tomonitor and direct the isolation and purification process.Cyanobacterial culture conditions, media and classification were asdescribed previously (Patterson, J. Phycol. 27: 530-536 (1991). Briefly,the cellular mass from a unialgal strain of Scylonema varium maintainedat the University of Hawaii at Manoa was harvested by filtration,freeze-dried and extracted with MeOH—CH₂Cl₂ (1:1) followed by H₂O.Bioassay data indicated that only the H₂O extract containedHIV-inhibitory activity.

A portion (10 g) of the aqueous extract was subjected to vacuum liquidchromatography on Bakerbond wide-pore C₄ media, eluting with a stepwisegradient of 0-100% methanol. A 1.0 g portion of the water/methanol 2:1v/v fraction (3.5 g total) was loaded on a Sephadex G-100 (5.5×19 cm)column and eluted with phosphate buffer (25 mM, pH 7.5) containing 0.4 MNaCl and 0.02% NaN₃. Final purification was achieved usingreversed-phase HPLC and eluting with a gradient of 0-60% acetonitrile in0.05% aqueous trifluoroacetic acid (TFA) in 40 min at a flow rate of 3mL/min, followed by 15 min of isocratic elution with 60% acetonitrile in0.05% aqueous TFA.

To facilitate sequence and structure determinations, disulfide bondswere reduced and alkylated by methods previously described (Bokesch etal., J. Nat. Prod. 64: 249-250 (2000)). The derivatized peptide waspurified by reversed-phase HPLC, using a gradient elution of 0.05%aqueous TFA for 40 min, then increasing to 60% acetonitrile in 0.05%aqueous TFA over 45 min. The S-(β-4-pyridylethyl)cysteine (PEC)derivative (250 μg) was subjected to endoproteinase Arg-C andendoproteinase Glu-C digestion per manufacturer's instructions at anenzyme/substrate ratio of 1:20. The cleaved peptide products werepurified by reversed-phase HPLC, using a gradient of 0.05% aqueous TFAfor 20 min, then increasing to 60% acetonitrile in 0.05% aqueous TFAover 100 min.

For disulfide bond determination a 1.0 mg sample of native, nonreducedscytovirin, 60 μl of 100 mM ammonium bicarbonate (pH 8.0), 6 μL ofacetonitrile and 6 μL of a 40 μM solution of trypsin in H₂O were added.The mixture was incubated at 37° C. for 16 hr, and then separated byreversed-phase HPLC, using a C₃ column (Zorbax) and eluting with alinear gradient from 0-100% acetonitrile in H₂O with 5% CH₃COOH (v/v) inthe mobile phase.

The scytovirin was isolated as described above in approximately 0.03%yield, and SDS-PAGE analysis showed only a single protein band, with arelative molecular mass of about 9 kDa. ESI-MS of the protein provided amolecular weight of 9,712.8 daltons.

Reduction and alkylation of the protein as described above with4-vinylpyridine generated the S-(β-4-pyridylethyl)cysteine (PEC)derivative, which gave an ESI-MS molecular weight of 10,774.3 daltons.This was consistent with the presence of 10 disulfide-linked cysteines.Amino acid analyses of scytovirin indicated that it contained twoglutamic acid and five arginine residues. Therefore, the alkylatedderivative of scytovirin was digested separately as described above withendoproteinases Arg-C and Glu-C to yield fragments amenable toN-terminal amino acid sequencing. The resulting eleven peptide fragmentswere sequenced, along with the intact PEC derivative, and analyzed byESI-MS to provide the entire sequence of scytovirin (FIG. 1).

Six fragments were obtained from the endoproteinase Arg-C digest. Thefragment consisting of residues 1-19 gave a molecular ion at m/z 2,096.0(calc. m/z 2,096.2), residues 20-30 gave m/z 1,405.3 (calc. m/z1,405.6), residues 31-43 gave m/z 1,530.4 (calc. m/z 1,530.7), residues44-67 gave m/z 2,686.8 (calc. m/z 2,686.9), residues 68-78 gave m/z1,378.2 (calc. m/z 1,378.6), and residues 79-95 gave m/z 1,765.7 (calc.m/z 1,766.0). These data fully supported the deduced amino acid sequenceof scytovirin.

Endoproteinase Glu-C cleaved peptide bonds C-terminally at glutamic acidand aspartic acid, producing five fragments, which also supported thededuced amino acid sequence. Fragments at m/z 1,217.8, 1,998.9, 3,591.9,1,986.2, and 2,050.0 corresponded to residues 1-10, 11-27, 28-58, 59-75,and 76-95, respectively, and provided overlapping confirmation of theamino acid sequence (FIG. 1). Therefore, it was shown that scytovirin isa 95 amino acid protein, of molecular weight 9,713 daltons, containingfive intrachain disulfide bonds.

To establish the locations of the intramolecular bonds, an aliquot ofnonreduced protein was treated with trypsin as described above and theresulting peptides were analyzed by ESI-MS. Peptide recognition software(http://sx102a.niddk.nih.gov/peptide) was used to determine thetheoretical disulfide bonded fragments. Two disulfide links wereunambiguously defined by the presence of the m/z fragments at 1318 and1553. The program gave the single match of Cys32-Cys38 for m/z 1318.Likewise, the m/z fragment at 1553 was in agreement with Cys80-Cys86.Two possible matches for m/z 2511 were seen, with the first involvingtwo disulfide links between the fragments consisting of amino acids20-30 and 3143. The second possibility consisted of one disulfide linkbetween amino acids 20-24 and 2543. The first option was not viablebecause of the already deduced disulfide bond between Cys32-Cys38, andbecause it was not possible to have two bonds between these fragments.The third disulfide bond was, thus, established as Cys20-Cys26.

A fragment at m/z 2,719 again gave two possible matches, one of whichwas two disulfide links between amino acids 68-78 and 79-95, and theother of which was, one disulfide link between amino acids 68-72 and73-95. As a Cys80-Cys86 bond had already been assigned, it was notpossible to have two disulfide links between the fragments, so aCys68-Cys74 bond was deduced. By process of elimination, the fifth bondwas assigned to Cys7-Cys55. This deduction was supported by a fragmentat m/z 3,851, which corresponded to links between amino acids 51-67 and1-19, and an additional fragment at m/z 3,158, which linked amino acids51-60 and 1-19. Thus, the disulfide linkage pattern was identified asCys20-Cys26, Cys32-Cys38, Cys68-Cys74, Cys80-Cys86, and Cys7-Cys55 asshown in FIG. 1.

Scytovirin shows strong internal sequence duplication. When amino acids1-48 and 49-95 are aligned, 36 residues (78%) share direct homology and2 (4%) represent conservative amino acid changes (FIG. 2). The bondsformed by the C20-26 and C32-38 disulfides correspond closely to thosedefined by the C68-74 and C80-86 disulfide links. These homologousregions, with two disulfide bridges linking cysteines located at sixresidue intervals, suggest the presence of two functional domains, whichare linked by the C7-55 bond.

Example 2

This example describes the production of anti-scytovirin antibodies.

A New Zealand white rabbit was immunized with 100 μg of scytovirin inFreund's complete adjuvant. Booster injections of 50 μg of scytovirin inFreund's incomplete adjuvant were administered on days 13, 29, 51, 64,100, and 195. On days 7, 21, 42, 63, 78, and 112, 10 mL of blood wereremoved from the rabbit. On day 112 the rabbit was sacrificed and bledout. The IgG fraction of the immune sera of the rabbit was isolated byprotein-A Sepharose affinity chromatography (Bio-Rad, Hercules, Calif.)according to the manufacturer's instructions. Reactivity of thepolyclonal antibodies for scytovirin was demonstrated by ELISA studieswith 1:100 to 1:1,000 of the rabbit immunoglobulin fractions.

Example 3

This example illustrates the evaluation of sequence homologies of ascytovirin with known proteins, and demonstrates that scytovirin doesnot have strong affinity for chitin.

A search of the BLAST database (Altschul et al., Nucleic Acid Res. 25:3389-3402 (1997)) for identification of protein sequence homologyindicated some apparent homology (55%) to a subsequence within a muchlarger cloned polypeptide from the multicellular green alga Volvoxcarteri (Amon et al., The Plant Cell 10: 781-789 (1998)) (FIG. 3). Thispolypeptide consists of three repeats of a 48-amino acid, chitin-bindingdomain separated by an extensin-like module from a cysteine proteasedomain. Of the 658 amino acids in the cloned polypeptide, scytovirinshowed homology to the chitin-binding domain, which consists of a commonstructural motif of 30-43 amino acids with glycines and cysteines atconserved positions. Although a large number of the cysteines areconserved, scytovirin has a different disulfide bonding pattern thanthat of the chitin-binding domains whose disulfide bridges have beendetermined (FIG. 4).

Because of the homology to the inner conserved core region ofchitin-binding proteins, the ability of scytovirin to bind to a chitinsubstrate was investigated. Scytovirin (100 μg) and wheat germagglutinin (WGA; 100 μg) each were dissolved in 900 μL ofphosphate-buffered saline (PBS) (pH 8.0) and applied to chitinmicrocolumns (8×10 mm). The samples were recycled three times over thecolumns, and eluted five times with 1 mL of PBS and once with 1.0 mL of0.1 M acetic acid. Samples were desalted and concentrated byreversed-phase HPLC, using a gradient of 0-60% acetonitrile in 0.05%aqueous TFA over 45 min at a flow rate of 3 mL/min, before beinganalyzed by SDS-PAGE. Scytovirin was present in the initial fractionthat was recycled through the column, while WGA was present only in thefraction eluted with low pH buffer. These results indicate that,although scytovirin contains a primary structural motif similar to thelectins and chitin-binding proteins, it does not exhibit strong bindingaffinity toward chitin.

Scytovirin showed a lower-scoring match (33%) to precursor proteins ofUrtica dioica agglutinin (IDA; Harata et al., J. Mol. Biol. 297: 673-681(2000)). Again, the homology was to the chitin-binding domains of UDA.Key amino acid residues involved in carbohydrate binding of UDA havebeen identified and include aromatic residues at positions 21, 23, and30, and a serine at position 19 (Does et al., Plant Mol. Biol. 39:335-347 (1999)). Scytovirin, in comparison, lacks aromatic residues atpositions 21 and 30, which may interfere with carbohydrate bindingactivity.

A conidiospore surface protein from Trichoderma harzianum (Horwitz,direct submission to BLAST; unpublished work; GI=4585623) and clonedantifreeze proteins from Dendroides canadensis (Andorfer et al., J.Insect Phys. 46: 365-372 (2000)) and Tenebrio molitor (Liou et al.,Biochemistry 38: 11415-11424 (1999)) showed lower scoring matches toscytovirin (31%, 27%, and 28%, respectively). The UDA proteins arecomprised of a signal peptide with two chitin-binding domains, a hingeregion and a carboxyl-terminal chitinase domain. The thermal hysterisis(antifreeze) proteins (THP), which showed homology, are a similar size(9 kDA), are Cys-, Thr-, and Ser-rich, are fully disulfide-bonded, andcontain repeated sequences of 12-amino acids.

Homology to the conidiospore surface protein and the THP is due mainlyto the conserved cysteines spaced at six-residue intervals. As there isno published data other than the sequence for the conidiospore protein,the function and importance of this spacing is unknown. The 12-aminoacid repeat found in the THP follows this six-residue cysteine spacingand, along with other key residues, is thought to be important for thestructural integrity and function of the antifreeze proteins.

Chitin-binding proteins with lectin properties are capable ofcross-linking GlcNAc- or NeuNAc-containing polymers due to the presenceof multiple chitin-binding domains. Since the envelope glycoprotein ofHIV is heavily glycosylated, HIV infectivity and virus-cell fusion maybe inhibited by lectins that are specific for the sugars present in thegp120 molecule. It has been shown that the D-mannose-specific lectin,concanavalin A (Lifson et al., J. Exp. Med. 164: 2101-2106 (1986)) doesblock HIV infectivity and virus-cell fusion, and the GlcNAc-specificlectins, myrianthin (Charan et al., J. Nat. Prod. 63: 1170-1174 (2000))and UDA-1 (Balzirini et al., Antiviral Res. 18: 191-207 (1992)), areinhibitors of HIV-induced cytopathicity.

Example 4

This example illustrates viral envelope molecular target interactions ofa scytovirin.

For these demonstrations, ELISA protocols were as follows. To determinethe affinities of scytovirin for a series of standard proteins, 100 ngeach of gp160, gp120, gp41, sCD4, bovine IgG, α-acid glycoprotein,aprotinin, HAS (human serum albumin), and BSA were subjected to an ELISAprotocol as previously described (O'Keefe et al., Mol. Pharmacol. 58:982-992 (2000)). Briefly, the proteins were bound to a 96-well plate,which was then rinsed with PBS containing 0.05% Tween 20 (TPBS) andblocked with BSA. Between each subsequent step, the plate was againrinsed with TPBS. The wells were incubated with 100 ng of scytovirin,followed by incubation with a 1:500 dilution of the anti-scytovirinrabbit polyclonal antibody preparation. The bound scytovirin wasdetermined by adding goat-anti-rabbit antibodies conjugated to alkalinephosphatase (Roche Molecular Biochemicals, Indianapolis, Ind.). Uponaddition of the alkaline phosphatase substrate buffer, absorbance wasmeasured at 405 nm for each well. Scytovirin interacted with gp160,gp120, and to a lesser degree, gp41, but not sCD4 or other referenceproteins, including bovine IgG, α-acid glycoprotein, aprotinin, HSA, andBSA.

Glycosylation-dependent binding of scytovirin to gp120 was examinedusing ELISA as above, with glycosylated and nonglycosylated gp120(HIV-1_(SF2) gp120) added to the 96-well plate and incubated with eightserial dilutions of scytovirin at a high concentration (100 ng/mL).Binding of scytovirin to gp120 was determined to beglycosylation-dependent.

To study the effect of monomeric and complex sugars on scytovirin andgp120 binding, ELISA plates were treated as above with the followingmodifications. The 96-well plates were first incubated with 100 ng ofgp120 and then treated with a preincubated (1 hr) 1:1 (v/v) mixture ofscytovirin/sugar to yield a final concentration of 0.005 mM scytovirinand 500 mM sugar per well. The monomeric sugars N-acetylgalactosanine,fucose, xylose, N-acetylglucosamine, mannose, glucose, and galactosewere tested as well as the complex oligosaccharides mannose 7, mannose8, mannose 9, a hybrid-type N-linked oligosaccharide, and an A3complex-type N-linked oligosaccharide.

Scytovirin was not inhibited from binding to gp120 byN-acetylgalactosamine, fucose, xylose, N-acetylglucosamine, mannose,glucose, and galactose. Unlike the lectins, scytovirin did not showspecificity for D-mannose, N-acetylglucosamine, orN-acetylgalactosamine, i.e., sugars associated with antiviral activity(Balzirini et al., Antiviral Res. 18: 191-207 (1992)). However, whentested against complex oligosaccharides, scytovirin-gp120 binding wasinhibited by oligomannose 8 and oligomannose 9, but not by oligomannose7, a hybrid-type N-linked oligosaccharide, or an A3 complex-typeN-linked oligosaccharide, consistent with results recently described forcyanovirin-N (Shenoy et al., J. Pharmacol. Exp. Ther. 297: 704-710(2001); and Bolmstedt et al., Mol. Pharmacol. 59: 949-954 (2001)),suggesting that scytovirin may interact preferentially with sites ongp120 comprising high-mannose oligosaccharides.

Although scytovirin has a lectin-like primary structure, it does notappear to belong to the chitin-binding or lectin class of proteins. Itdoes not bind chitin, does not have the same disulfide bonding patternas the chitin-binding domains determined thus far, and lacks some of thekey aromatic amino acid residues involved in carbohydrate binding.

Example 5

This example illustrates antiviral activity, in particular anti-HIVactivity, of a scytovirin.

An XTT-tetrazolium based assay was used to determine the anti-HIVactivity of scytovirin on acute HIV-1 infection in CEM-SS cells aspreviously described (Gulakowski et al., J. Virol. Methods 33: 87-100(1991)). The effects of scytovirin on pretreatment of CEM-SS cells andHIV-1_(RF), delayed addition to HIV-1_(RF) infected cells, and cell-cellfusion were studied using methods described previously (O'Keefe et al.,Eur. J. Biochem. 245: 47-53 (1997)).

Antiviral assays used to study the activities of laboratory strains andprimary isolates of virus have been previously published (Buckheit,Antiviral Res. 21: 247-265 (1993)). The low passage HIV-I pediatricisolate ROJO was derived as previously described (Buckheit et al., AIDSRes. Hum. Retroviruses 10: 1497-1506 (1991)). Peripheral bloodmononuclear cells (PBMC) and macrophages were isolated from hepatitisand HIV sero-negative donors following Ficoll-Hypaque centrifugation asdescribed elsewhere (Gartner, Techniques in HIV Research, Aldovini, A.and Walker, B., Eds.; Stockton Press: New York (1994), pp 59-63).

Attachment and additional fusion assays were performed as previouslydescribed with the modifications listed below. Descriptions and sourcesof the cell lines have been previously published (Buckheit et al., AIDSRes. Hum. Retroviruses 10: 1497-1506 (1994)). HL2/3 and HeLa CD4 LTRβ-gal cell lines were maintained in Dulbecco's Minimal Essential Medium(DMEM) with 10% fetal bovine serum, penicillin (100 U/mL), streptomycin(100 μg/mL) and L-glutamine (2 mM). HeLa CD4 LTR β-gal cell lines werealso supplemented with G418 (200 μg/mL) and hygromycin B (100 μg/mL).Following the interaction of HIV-1_(IIIB) with HeLa CD4 LTR β-gal cells(attachment assay) or the coculture of HeLa CD4 LTR β-gal and HL2/3cells (fusion assay), viral replication was detected bychemiluminescence using a single-step lysis and detection method (TropixGal-screen™, Bedford, Mass.). Viral binding to HeLa CD4 LTR β-gal cellswas detected as cell-associated p24 antigen, following a 1 hr adsorptionof virus and vigorous washing to remove unbound virus. Chicago Sky Blue,a polysulfonic acid dye inhibitor of HIV attachment and fusion, was usedas a positive control for all assays (Clanton et al., J. Acquir. ImmuneDefic. Syndr. 5: 771-781 (1992)).

Scytovirin showed comparable activity against the T-tropic laboratorystrain HIV-1_(RF) in CEM-SS cells and primary isolate ROJO in PBMC'swith EC₅₀ values of 0.3 nM and 7 nM. Scytovirin was also active againstthe M-tropic primary isolate Ba-L in macrophages, with an EC₅₀ value of22 nM. Delayed addition experiments showed that scytovirin had to bepresent within the first 8 hr of viral infection for maximum antiviralactivity, consistent with a primary effect of scytovirin on thevirus/cell attachment and/or fusion process. Cell- andvirus-pretreatment and delayed addition studies of scytovirin suggestedthat it must be continually present early in the viral life cycle to bemaximally protective.

Cocultivation of uninfected and chronically infected CEM-SS cells withscytovirin caused a concentration-dependent inhibition of cell-cellfusion. Additional binding and fusion inhibition assays using β-galindicator cells gave similar results. Scytovirin inhibited fusion ofCD4+ β-gal cells with HL2/3 cells as well as the cell-free HIV-1_(IIIB)fusion and infection of β-gal cells in a concentration-dependent manner.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

1. An isolated or purified antiviral protein comprising the amino acidsequence of SEQ ID NO:
 1. 2. A fusion protein comprising the isolated orpurified antiviral protein of claim
 1. 3. The fusion protein of claim 2,which comprises albumin.
 4. An isolated or purified nucleic acidcomprising a nucleotide sequence encoding the fusion protein of claim 2,optionally in the form of a vector.
 5. An isolated cell comprising theisolated or purified nucleic acid of claim
 4. 6. The isolated cell ofclaim 5, which is a bacterium or a yeast.
 7. The isolated cell of claim6, wherein the bacterium is a lactobacillus.
 8. A conjugate comprisingthe isolated or purified antiviral protein of claim 1 and at least oneeffector component.
 9. The conjugate of claim 8, wherein the at leastone effector component can be the same or different and is selected fromthe group consisting of polyethylene glycol, dextran, a toxin, animmunological reagent, an antiviral agent, and a solid support matrix.10. A composition comprising (i) at least one isolated or purifiedantiviral protein of claim 1, a fusion protein thereof, and a conjugatethereof and (ii) a carrier, excipient or adjuvant therefor.
 11. Thecomposition of claim 10, wherein (i) the composition is present in anantiviral effective amount and (ii) the composition is pharmaceuticallyacceptable.
 12. An isolated or purified nucleic acid comprising anucleotide sequence encoding the protein of claim 1, optionally in theform of a vector.
 13. An isolated cell comprising the isolated orpurified nucleic acid of claim
 12. 14. The isolated cell of claim 13,which is a bacterium or a yeast.
 15. The isolated cell of claim 14,wherein the bacterium is lactobacillus.
 16. A composition comprising (i)the isolated or purified nucleic acid of claim 12, optionally as part ofan encoded fusion protein, and (ii) a carrier, excipient or adjuvanttherefor.
 17. The composition of claim 16, wherein (i) is present in anantiviral effective amount and the composition is pharmaceuticallyacceptable.
 18. A method of inhibiting a viral infection of a host,which method comprises administering a viral infection-inhibiting amountof at least one of the following: i. an isolated or purified antiviralprotein of claim 1, ii. a fusion protein of (i), iii. a conjugatecomprising (i) and at least one effector component, iv. a compositioncomprising one or more of (i)-(iii), v. an isolated or purified nucleicacid comprising a nucleotide sequence encoding the amino acid sequenceof claim 1, optionally in the form of a vector, vi. an isolated orpurified nucleic acid comprising a nucleotide sequence encoding a fusionprotein of (v), optionally in the form of a vector, vii. a compositioncomprising one or more of (v)-(vi), and an isolated cell comprising (v)or (vi), wherein the viral infection is caused by a virus having aglycoprotein comprising a high-mannose oligosaccharide as a coatprotein, which method optionally further comprises the prior,simultaneous or subsequent administration, by the same route or adifferent route, of an antiviral agent or another agent that isefficacious in inhibiting the viral infection, whereupon the viralinfection is inhibited.
 19. The method of claim 18, wherein the virus isan immunodeficiency virus.
 20. The method of claim 19, wherein thefusion protein comprises albumin.
 21. The method of claim 19, whereinthe at least one effector component can be the same or different and isselected from the group consisting of polyethylene glycol, dextran, atoxin, an immunological reagent, an antiviral agent, and a solid supportmatrix.
 22. The method of claim 19, wherein the isolated cell is a cellfrom the host, which had been previously isolated and contacted with (v)or (vi).
 23. The method of claim 19, wherein the isolated cell is a cellfrom a homologous host.
 24. The method of claim 19, wherein the isolatedcell is a nonpathogenic bacterium or a yeast.
 25. The method of claim18, wherein the host is a human and the immunodeficiency virus is humanimmunodeficiency virus (HIV).
 26. The method of claim 25, wherein thefusion protein comprises albumin.
 27. The method of claim 25, whereinthe at least one effector component can be the same or different and isselected from the group consisting of polyethylene glycol, dextran, atoxin, an immunological reagent, an antiviral agent, and a solid supportmatrix.
 28. The method of claim 25, wherein the isolated cell is a cellfrom the host, which had been previously isolated and contacted with (v)or (vi).
 29. The method of claim 25, wherein the isolated cell is a cellfrom a homologous host.
 30. The method of claim 25, wherein the isolatedcell is a nonpathogenic bacterium or a yeast.
 31. The method of claim18, wherein the fusion protein comprises albumin.
 32. The method ofclaim 18, wherein the at least one effector component can be the same ordifferent and is selected from the group consisting of polyethyleneglycol, dextran, a toxin, an immunological reagent, an antiviral agent,and a solid support matrix.
 33. The method of claim 18, wherein theisolated cell is a cell from the host, which had been previouslyisolated and contacted with (v) or (vi).
 34. The method of claim 18,wherein the isolated cell is a cell from a homologous host.
 35. Themethod of claim 18, wherein the isolated cell is a nonpathogenicbacterium or a yeast.
 36. The method of claim 35, wherein thenonpathogenic bacterium is a lactobacillus.
 37. The method of claim 18,wherein the viral infection is an influenza infection.
 38. The method ofclaim 18, wherein the viral infection is an Ebola infection.
 39. Themethod of claim 18, wherein the host is an avian host.
 40. The method ofclaim 18, wherein at least one of (i)-(vii) is administered nasally, byinhalation, or by parenteral administration.
 41. A method of inhibitinga virus in a biological sample or in/on an inanimate object, whichmethod comprises contacting the biological sample or the inanimateobject with a viral-inhibiting amount of at least one of the following:i. an isolated or purified antiviral protein of claim 1, ii. a fusionprotein of (i), iii. a conjugate comprising (i) and at least oneeffector component, iv. a composition comprising one or more of(i)-(iii), wherein the viral infection is caused by a virus having aglycoprotein comprising a high-mannose oligosaccharide as a coatprotein, which method optionally further comprises the prior,simultaneous or subsequent contacting, in the same manner or in adifferent manner, of the biological sample or inanimate object with anantiviral agent or another agent that is efficacious in inhibiting thevirus, whereupon the virus is inhibited.
 42. The method of claim 41,wherein the biological sample is blood, a blood product, cells, atissue, an organ, sperm, a vaccine formulation, or a bodily fluid. 43.The method of claim 41, wherein the inanimate object is a solution, amedical supply, or a medical equipment.
 44. The method of claim 41,wherein the fusion protein comprises albumin.
 45. The method of claim41, wherein the at least one effector component can be the same ordifferent and is selected from the group consisting of polyethyleneglycol, dextran, a toxin, an immunological reagent, an antiviral agent,and a solid support matrix.
 46. An isolated or purified antiviralprotein consisting essentially of the amino acid sequence of SEQ ID NO:1, an amino acid sequence that is about 90% or more identical to SEQ IDNO: 1or an amino acid sequence that is about 90% or more homologous toSEQ ID NO: 1, which has been isolated or purified from Scytonema varium.